Methods and compositions for treating aging-associated conditions

ABSTRACT

Methods of treating an adult mammal for an aging-associated condition are provided. Aspects of the methods include enhancing a TIMP activity, e.g., a TIMP2 activity, in the mammal in a manner sufficient to treat the adult mammal for the aging-associated condition. Also provided are compositions for use in practicing methods of the invention. A variety of aging-associated conditions may be treated by practice of the methods, which conditions include cognitive impairments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. 371 national phase entry of InternationalPatent Application No. PCT/US2016/036032, filed Jun. 6, 2016, whichclaims priority to the filing date of the U.S. Provisional PatentApplication Ser. No. 62/175,981 filed Jun. 15, 2015, the disclosure ofwhich applications are incorporated herein by reference in theirentireties.

GOVERNMENT RIGHTS

This invention was made with Government support under contract AG045034awarded by the National Institutes of Health. The Government has certainrights in the invention.

INTRODUCTION

Aging in an organism is accompanied by an accumulation of changes overtime. In the nervous system, aging is accompanied by structural andneurophysiological changes that drive cognitive decline andsusceptibility to degenerative disorders in healthy individuals. (Heeden& Gabrieli, “Insights into the ageing mind: a view from cognitiveneuroscience,” Nat. Rev. Neurosci. (2004) 5: 87-96; Raz et al.,“Neuroanatomical correlates of cognitive aging: evidence from structuralmagnetic resonance imaging,” Neuropsychology (1998) 12:95-114; Mattson &Magnus, “Ageing and neuronal vulnerability,” Nat. Rev. Neurosci. (2006)7: 278-294; and Rapp & Heindel, “Memory systems in normal andpathological aging,” Curr. Opin. Neurol. (1994) 7:294-298). Included inthese changes are synapse loss and the loss of neuronal function thatresults. Thus, although significant neuronal death is typically notobserved during the natural aging process, neurons in the aging brainare vulnerable to sub-lethal age-related alterations in structure,synaptic integrity, and molecular processing at the synapse, all ofwhich impair cognitive function.

In addition to the normal synapse loss during natural aging, synapseloss is an early pathological event common to many neurodegenerativeconditions, and is the best correlate to the neuronal and cognitiveimpairment associated with these conditions. Indeed, aging remains thesingle most dominant risk factor for dementia-related neurodegenerativediseases such as Alzheimer's disease (AD) (Bishop et al., “Neuralmechanisms of ageing and cognitive decline,” Nature (2010) 464: 529-535(2010); Heeden & Gabrieli, “Insights into the ageing mind: a view fromcognitive neuroscience,” Nat. Rev. Neurosci. (2004) 5:87-96; Mattson &Magnus, “Ageing and neuronal vulnerability,” Nat. Rev. Neurosci. (2006)7:278-294).

As the human lifespan increases, a greater fraction of the populationsuffers from aging-associated cognitive impairments, making it crucialto elucidate means by which to maintain cognitive integrity byprotecting against, or even counteracting, the effects of aging (Hebertet al., “Alzheimer disease in the US population: prevalence estimatesusing the 2000 census,” Arch. Neurol. (2003) 60:1119-1122; Bishop etal., “Neural mechanisms of ageing and cognitive decline,” Nature (2010)464:529-535).

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is a member of a groupof specific inhibitors of matrix metalloproteinases. The proteins theseinhibitors regulate, matrix metalloproteinases (MMPs), play a role inseveral physiological processes including growth, wound healing, tissuerepair, and cellular development and homeostasis. Broad categories ofMMPs consist of collagenases, gelatinases, stromelysins, matrilysins,membrane-type MMPs (MT-MMPs), and others. These enzymes must beprecisely regulated as the loss of control over MMP activity may resultin arthritis, cancer, atherosclerosis, aneurysms, nephritis, tissueulcers, fibrosis, and other tissue damage (Visse and Nagase, “MatrixMetalloproteinases and Tissue Inhibitors of Metalloproteinases,”Circulation Research (2003) 92: 827-39). Four TIMPs (1-4) have beenidentified in vertebrates.

SUMMARY

Methods of treating an adult mammal for an aging-associated conditionare provided. Aspects of the methods include enhancing a TIMP activity,e.g., a TIMP2 activity, in the mammal in a manner sufficient to treatthe adult mammal for the aging-associated condition. Also provided arecompositions for use in practicing methods of the invention. A varietyof aging-associated conditions may be treated by practice of themethods, which conditions include cognitive impairments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the results on brain slices isolated from aged wildtypemice that were treated with recombinant TIMP2 (i.p., 50 μg/kg)(bottom)or a control (top). FIG. 1B provides quantification of the maintenancephase of the PSA shown in FIG. 1A.

FIG. 2 shows the results of anti-TIMP2 antibody treatment on objectdiscrimination.

DETAILED DESCRIPTION

Methods of treating an adult mammal for an aging-associated conditionare provided. Aspects of the methods include enhancing TIMP activity,e.g., a TIMP2 activity, in the mammal in a manner sufficient to treatthe adult mammal for the aging-associated condition. Also provided arecompositions for use in practicing methods of the invention. A varietyof aging-associated conditions may be treated by practice of themethods, which conditions include cognitive impairments.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to a particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof,e.g., polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Methods

As summarized above, aspects of the invention include methods oftreating an aging-associated condition in an adult mammal. Theaging-associated condition may manifest in a number of different ways,e.g., as aging-associated cognitive impairment and/or physiologicalimpairment, e.g., in the form of damage to central or peripheral organsof the body, such as but not limited to: cell injury, tissue damage,organ dysfunction, aging-associated lifespan shortening andcarcinogenesis, where specific organs and tissues of interest include,but are not limited to skin, neuron, muscle, pancreas, brain, kidney,lung, stomach, intestine, spleen, heart, adipose tissue, testes, ovary,uterus, liver and bone; in the form of decreased neural plasticity, etc.

In some embodiments, the aging-associated condition is anaging-associated impairment in cognitive ability in an individual, i.e.,an aging-associated cognitive impairment. By cognitive ability, or“cognition”, it is meant the mental processes that include attention andconcentration, learning complex tasks and concepts, memory (acquiring,retaining, and retrieving new information in the short and/or longterm), information processing (dealing with information gathered by thefive senses), visuospatial function (visual perception, depthperception, using mental imagery, copying drawings, constructing objectsor shapes), producing and understanding language, verbal fluency(word-finding), solving problems, making decisions, and executivefunctions (planning and prioritizing). By “cognitive decline”, it ismeant a progressive decrease in one or more of these abilities, e.g., adecline in memory, language, thinking, judgment, etc. By “an impairmentin cognitive ability” and “cognitive impairment”, it is meant areduction in cognitive ability relative to a healthy individual, e.g.,an age-matched healthy individual, or relative to the ability of theindividual at an earlier point in time, e.g., 2 weeks, 1 month, 2months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years ormore previously. Aging-associated cognitive impairments includeimpairments in cognitive ability that are typically associated withaging, including, for example, cognitive impairment associated with thenatural aging process, e.g., mild cognitive impairment (M.C.I.); andcognitive impairment associated with an aging-associated disorder, thatis, a disorder that is seen with increasing frequency with increasingsenescence, e.g., a neurodegenerative condition such as Alzheimer'sdisease, Parkinson's disease, frontotemporal dementia, Huntington'sdisease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma,muscular dystrophy, vascular dementia, and the like.

By “treatment” it is meant that at least an amelioration of one or moresymptoms associated with an aging-associated condition afflicting theadult mammal is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g., asymptom associated with the impairment being treated. As such, treatmentalso includes situations where a pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g., terminated, such that the adult mammalno longer suffers from the impairment, or at least the symptoms thatcharacterize the impairment. In some instances, “treatment”, “treating”and the like refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete cure for a diseaseand/or adverse effect attributable to the disease. “Treatment” may beany treatment of a disease in a mammal, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, i.e., arresting its development; or (c) relieving the disease,i.e., causing regression of the disease. Treatment may result in avariety of different physical manifestations, e.g., modulation in geneexpression, increased synaptic efficacy, increased neurogenesis,rejuvenation of tissue or organs, etc. Treatment of ongoing disease,where the treatment stabilizes or reduces the undesirable clinicalsymptoms of the patient, occurs in some embodiments. Such treatment maybe performed prior to complete loss of function in the affected tissues.The subject therapy may be administered prior to the symptomatic stateof the disease, during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease.

In some instances where the aging-associated condition isaging-associated cognitive decline, treatment by methods of the presentdisclosure slows, or reduces, the progression of aging-associatedcognitive decline. In other words, cognitive abilities in the individualdecline more slowly, if at all, following treatment by the disclosedmethods than prior to or in the absence of treatment by the disclosedmethods. In some instances, treatment by methods of the presentdisclosure stabilizes the cognitive abilities of an individual. Forexample, the progression of cognitive decline in an individual sufferingfrom aging-associated cognitive decline is halted following treatment bythe disclosed methods. As another example, cognitive decline in anindividual, e.g., an individual 40 years old or older, that is projectedto suffer from aging-associated cognitive decline, is preventedfollowing treatment by the disclosed methods. In other words, no(further) cognitive impairment is observed. In some instances, treatmentby methods of the present disclosure reduces, or reverses, cognitiveimpairment, e.g., as observed by improving cognitive abilities in anindividual suffering from aging-associated cognitive decline. In otherwords, the cognitive abilities of the individual suffering fromaging-associated cognitive decline following treatment by the disclosedmethods are better than they were prior to treatment by the disclosedmethods, i.e., they improve upon treatment. In some instances, treatmentby methods of the present disclosure abrogates cognitive impairment. Inother words, the cognitive abilities of the individual suffering fromaging-associated cognitive decline are restored, e.g., to their levelwhen the individual was about 40 years old or less, following treatmentby the disclosed methods, e.g., as evidenced by improved cognitiveabilities in an individual suffering from aging-associated cognitivedecline.

In some instances, treatment of an adult mammal in accordance with themethods results in a change in a central organ, e.g., a central nervoussystem organ, such as the brain, spinal cord, etc., where the change maymanifest in a number of different ways, e.g., as described in greaterdetail below, including but not limited to molecular, structural and/orfunctional, e.g., in the form of enhanced synaptic plasticity. In someinstances, treatment of a subject in accordance with the methods resultsin a change in a peripheral organ, such as liver, muscle, heart, blood,etc., where the change may manifest in a number of different ways, e.g.,as described in greater detail below.

As summarized above, methods described herein are methods of treating anaging-associated condition, e.g., as described above, in an adultmammal. By adult mammal is meant a mammal that has reached maturity,i.e., that is fully developed. As such, adult mammals are not juvenile.Mammalian species that may be treated with the present methods includecanines and felines; equines; bovines; ovines; etc., and primates,including humans. The subject methods, compositions, and reagents mayalso be applied to animal models, including small mammals, e.g., murine,lagomorpha, etc., for example, in experimental investigations. Thediscussion below will focus on the application of the subject methods,compositions, reagents, devices and kits to humans, but it will beunderstood by the ordinarily skilled artisan that such descriptions canbe readily modified to other mammals of interest based on the knowledgein the art.

The age of the adult mammal may vary, depending on the type of mammalthat is being treated. Where the adult mammal is a human, the age of thehuman is generally 18 years or older. In some instances, the adultmammal is an individual suffering from or at risk of suffering from anaging-associated impairment, such as an aging-associated cognitiveimpairment, where the adult mammal may be one that has been determined,e.g., in the form of receiving a diagnosis, to be suffering from or atrisk of suffering from an aging-associated impairment, such as anaging-associated cognitive impairment. The phrase “an individualsuffering from or at risk of suffering from an aging-associatedcognitive impairment” refers to an individual that is about 50 years oldor older, e.g., 60 years old or older, 70 years old or older, 80 yearsold or older, and sometimes no older than 100 years old, such as 90years old, i.e., between the ages of about 50 and 100, e.g., 50, 55, 60,65, 70, 75, 80, 85 or about 90 years old. The individual may suffer froman aging associated condition, e.g., cognitive impairment, associatedwith the natural aging process, e.g., M.C.I. Alternatively, theindividual may be 50 years old or older, e.g., 60 years old or older, 70years old or older, 80 years old or older, 90 years old or older, andsometimes no older than 100 years old, i.e., between the ages of about50 and 100, e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100years old, and has not yet begun to show symptoms of an aging associatedcondition, e.g., cognitive impairment. In yet other embodiments, theindividual may be of any age where the individual is suffering from acognitive impairment due to an aging-associated disease, e.g.,Alzheimer's disease, Parkinson's disease, frontotemporal dementia,Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis,glaucoma, muscular dystrophy, dementia, and the like. In some instances,the individual is an individual of any age that has been diagnosed withan aging-associated disease that is typically accompanied by cognitiveimpairment, e.g., Alzheimer's disease, Parkinson's disease,frontotemporal dementia, progressive supranuclear palsy, Huntington'sdisease, amyotrophic lateral sclerosis, spinal muscular atrophy,multiple sclerosis, multi-system atrophy, glaucoma, ataxias, musculardystrophy, dementia, and the like, where the individual has not yetbegun to show symptoms of cognitive impairment.

As summarized above, aspects of the methods include enhancing a TIMPactivity, e.g., a systemic TIMP activity, in the mammal in a mannersufficient to treat the adult mammal for the aging-associated condition.By enhancing a TIMP activity is meant increasing one or more target TIMPactivities in the subject. In some instances, the TIMP activity that isenhanced is a systemic TIMP activity, by which is meant a TIMP activityin the circulatory system of the mammal. The magnitude of the increasemay vary, where in some instances the magnitude of the increase is2-fold or greater, such as 5-fold or greater, including 10-fold orgreater, e.g., 15-fold or greater, 20-fold or greater, 25-fold orgreater (as compared to a suitable control). The TIMP activity that isincreased by practice of the methods is a TIMP mediated process that isbeneficial in treating an aging associated condition. In other words,the TIMP activity that is enhanced is one that results in treatment,e.g., as described above, of the subject for the aging associatedcondition.

The target TIMP activity that is enhanced may vary. In some instances,the target TIMP activity is a TIMP2 activity, i.e., an activityexhibited by a TIMP2 protein. As such, by TIMP2 activity is meant anactivity of interest of a TIMP2 protein, i.e., an activity that resultsin treatment of an aging-associated condition, e.g., as described above.Of interest are mammalian TIMP2 proteins, such as but not limited to:primate, e.g., human, canine, feline, equine, bovine, ovine, murine,lagomorpha, etc. The sequence of human TIMP2 is:

(SEQ ID NO: 01)         10         20         30         40         50MGAAARTLRL ALGLLLLATL LRPADACSCS PVHPQQAFCN ADVVIRAKAV        60         70         80         90        100SEKEVDSGND IYGNPIKRIQ YEIKQIKMFK GPEKDIEFIY TAPSSAVCGV       110        120        130        140        150SLDVGGKKEY LIAGKAEGDG KMHITLCDFI VPWDTLSTTQ KKSLNHRYQM       160        170        180        190        200GCECKITRCP MIPCYISSPD ECLWMDWVTE KNINGHQAKF FACIKRSDGS       210        220 CAWYRGAAPP KQEFLDIEDP

In some instances, the target TIMP activity is a TIMP1 activity, i.e.,an activity exhibited by a TIMP1 protein. As such, by TIMP1 activity ismeant an activity of interest of a TIMP1 protein, i.e., an activity thatresults in treatment of an aging-associated condition, e.g., asdescribed above. Of interest are mammalian TIMP1 proteins, such as butnot limited to: primate, e.g., human, canine, feline, equine, bovine,ovine, murine, lagomorpha, etc. The sequence of human TIMP1 is:

(SEQ ID NO: 02)         10         20         30         40         50MAPFEPLASG ILLLLWLIAP SRACTCVPPH PQTAFCNSDL VIRAKFVGTP        60         70         80         90        100EVNQTTLYQR YEIKMTKMYK GFQALGDAAD IRFVYTPAME SVCGYFHRSH       110        120        130        140        150NRSEEFLIAG KLQDGLLNIT TCSFVAPWNS LSLAQRRGFT KTYTVGCEEC       160        170        180        190        200TVFPCLSIPC KLQSGTHCLW TDQLLQGSEK GFQSRHLACL PREPGLCTWQ SLRSQIA 

In some instances, the target TIMP activity is a TIMP3 activity, i.e.,an activity exhibited by a TIMP3 protein. As such, by TIMP3 activity ismeant an activity of interest of a TIMP3 protein, i.e., an activity thatresults in treatment of an aging-associated condition, e.g., asdescribed above. Of interest are mammalian TIMP3 proteins, such as butnot limited to: primate, e.g., human, canine, feline, equine, bovine,ovine, murine, lagomorpha, etc. The sequence of human TIMP3 is:

(SEQ ID NO: 03)         10         20         30         40         50MTPWLGLIVL LGSWSLGDWG AEACTCSPSH PQDAFCNSDI VIRAKVVGKK        60         70         80         90        100LVKEGPFGTL VYTIKQMKMY RGFTKMPHVQ YIHTEASESL CGLKLEVNKY       110        120        130        140        150QYLLTGRVYD GKMYTGLCNF VERWDQLTLS QRKGLNYRYH LGCNCKIKSC       160        170        180        190        200YYLPCFVTSK NECLWTDMLS NFGYPGYQSK HYACIRQKGG YCSWYRGWAP        210PDKSIINATD P

In some instances, the target TIMP activity is a TIMP4 activity, i.e.,an activity exhibited by a TIMP4 protein. As such, by TIMP2 activity ismeant an activity of interest of a TIMP4 protein, i.e., an activity thatresults in treatment of an aging-associated condition, e.g., asdescribed above. Of interest are mammalian TIMP4 proteins, such as butnot limited to: primate, e.g., human, canine, feline, equine, bovine,ovine, murine, lagomorpha, etc. The sequence of human TIMP4 is:

(SEQ ID NO: 04)         10         20         30         40         50MPGSPRPAPS WVLLLRLLAL LRPPGLGEAC SCAPAHPQQH ICHSALVIRA        60         70         80         90        100KISSEKVVPA SADPADTEKM LRYEIKQIKM FKGFEKVEDV QYIYTPFDSS       110        120        130        140        150LCGVKLEANS QKQYLLTGQV LSDGKVFIHL CNYIEPWEDL SLVQRESLNH       160        170        180        190        200HYHLNCGCQI TTCYTVPCTI SAPNECLWTD WLLERKLYGY QAQHYVCMKH       210        220 VDGTCSWYRG HLPLRKEFVD IVQP

The target TIMP activity or activities of interest may be enhanced usingany convenient protocol. In some instances, the target TIMP activity isenhanced by increasing a systemic level of a TIMP active agent in themammal. By systemic level is meant the level (e.g., concentration oramount) of the TIMP active agent in the circulatory system of themammal. The magnitude of the increase may vary, where in some instancesthe magnitude of the increase is 2-fold or greater, such as 5-fold orgreater, including 10-fold or greater, e.g., 15-fold or greater, 20-foldor greater, 25-fold or greater (as compared to a suitable control).

In these embodiments, the systemic level of the TIMP active agent ofinterest may be increased using any convenient protocol. In someinstances, the systemic level is increased by administering a TIMPactive agent to the subject. In such instances, the TIMP active agentmay vary. TIMP active agents that may be employed in these embodimentsof the invention include TIMP polypeptides and nucleic acids encodingthe same.

TIMP polypeptides are polypeptides that, upon administration to asubject, exhibit the desired TIMP aging associated condition treatmentactivity, e.g., as described above. The term “polypeptide” as usedherein refers to full-length proteins as well as portions or fragmentsthereof which exhibit the desired TIMP activity. Also included in thisterm are variations of the naturally occurring proteins, where suchvariations are homologous or substantially similar to the naturallyoccurring protein, as described in greater detail below, be thenaturally occurring protein the human protein, mouse protein, or proteinfrom some other species which naturally expresses a TIMP protein. In thefollowing description, the term TIMP is used to refer not only to thehuman form of a TIMP protein, but also to homologs thereof expressed innon-human species.

TIMP polypeptides of interest may vary in terms of amino acid sequencelength and molecular weight. In some instances, the TIMP polypeptidesrange in length from 175 to 350, such as from 200 to 250 and includingfrom about 200 to 225 amino acid residues, and have a projectedmolecular weight based solely on the number of amino acid residues inthe protein and assuming an average molecular weight of 110 Daltons thatranges from 19 to 39 kDa, such as 22 to 28 kDa, including 22 to 25 kDa,where the actual molecular weight may vary depending on the amount ofglycosylation of the protein and the apparent molecular weight may beconsiderably less because of SDS binding on gels. TIMP polypeptides asdescribed herein may be obtained from naturally sources, e.g., viapurification techniques, chemically synthesized or produced usingrecombinant protocols, as desired.

In some instances, the TIMP polypeptide that is administered to thesubject is a human TIMP2 protein, where the human TIMP2 protein has anamino acid sequence that comprises a region substantially the same as oridentical to the sequence appearing as SEQ ID NO:01. By substantiallythe same as is meant a protein having a region with a sequence that is60% or greater, such as 75% or greater, such as 90% or greater andincluding 98% or greater sequence identity with the sequence of SED IDNO:01, as determined by BLAST using default settings. In some instances,the TIMP polypeptide that is administered to the subject is a humanTIMP1 protein, where the human TIMP1 protein has an amino acid sequencethat comprises a region substantially the same as or identical to thesequence appearing as SEQ ID NO:02. By substantially the same as ismeant a protein having a region with a sequence that is 60% or greater,such as 75% or greater, such as 90% or greater and including 98% orgreater sequence identity with the sequence of SED ID NO:02, asdetermined by BLAST using default settings. In some instances, the TIMPpolypeptide that is administered to the subject is a human TIMP3protein, where the human TIMP3 protein has an amino acid sequence thatcomprises a region substantially the same as or identical to thesequence appearing as SEQ ID NO:03. By substantially the same as ismeant a protein having a region with a sequence that is 60% or greater,such as 75% or greater, such as 90% or greater and including 98% orgreater sequence identity with the sequence of SED ID NO:03, asdetermined by BLAST using default settings. In some instances, the TIMPpolypeptide that is administered to the subject is a human TIMP4protein, where the human TIMP4 protein has an amino acid sequence thatcomprises a region substantially the same as or identical to thesequence appearing as SEQ ID NO:04. By substantially the same as ismeant a protein having a region with a sequence that is 60% or greater,such as 75% or greater, such as 90% or greater and including 98% orgreater sequence identity with the sequence of SED ID NO:04, asdetermined by BLAST using default settings.

In addition to the specific TIMP proteins described above, homologs orproteins (or fragments thereof) from other species, e.g., other animalspecies, may also be employed in embodiments of the methods, where suchhomologs or proteins may be from a variety of different types ofspecies, including animals, such as mammals, e.g., rodents, such asmice, rats; domestic animals, e.g., horse, cow, dog, cat; etc. Byhomolog is meant a protein having 35% or more, such as 40% and more andincluding 60% or more amino acid sequence identity to the specific TIMPproteins as identified in SEQ ID NOS: 01 to 04, where sequence identityis determined using BLAST at default settings.

In addition to the naturally occurring TIMP proteins, e.g., as describedabove, TIMP polypeptides that vary from the naturally occurring TIMPproteins may also be employed in practicing methods of the invention.Different variations may be present, including but not limited tosubstitution, insertion and/or deletion mutations, as well as othertypes of non-amino acid sequence variations, e.g., as illustrated below.TIMP polypeptides that may be employed include proteins having an aminoacid sequence encoded by an open reading frame (ORF) of a TIMP gene,including the full length TIMP protein and fragments thereof, such asbiologically active fragments and/or fragments corresponding tofunctional domains; and including fusions of the subject polypeptides toother proteins or parts thereof. Fragments of interest may vary inlength, and in some instances are 10 aa or longer, such as 50 aa orlonger, and including 100 aa or longer, and in some instances do notexceed 150 aa in length, where a given fragment will have a stretch ofamino acids that is substantially the same as or identical to asubsequence found in any of SEQ ID NOS:1 to 4; where the subsequence mayvary in length and in some instances is 10 aa or longer, such as 15 aaor longer, up to 50 aa or even longer.

In some instances, TIMP polypeptides employed in methods of inventioninclude or more modifications. Modifications that may be present mayvary, and include but are not limited to: amide bond substitutions,amino acid substitutions, including of cysteine residues/analogues,cyclization, pegylation, etc. Examples of modifications that may befound in TIMP polypeptides employed in methods of the invention are nowreviewed in greater detail.

In some cases, TIMP polypeptides include one or more linkages other thanpeptide bonds, e.g., at least two adjacent amino acids are joined via alinkage other than an amide bond. For example, in order to reduce oreliminate undesired proteolysis or other means of degradation, and/or toincrease serum stability, and/or to restrict or increase conformationalflexibility, one or more amide bonds within the backbone of a TIMPpolypeptide can be substituted. In another example, one or more amidelinkages (—CO—NH—) in a TIMP polypeptide can be replaced with a linkagewhich is an isostere of an amide linkage, such as —CH₂NH—, —CH₂S—,—CH₂CH₂—, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂— or —CH₂SO—. Oneor more amide linkages in a TIMP polypeptide can also be replaced by,for example, a reduced isostere pseudopeptide bond.

One or more amino acid substitutions can be made in a TIMP polypeptide.The following are non-limiting examples: a) substitution ofalkyl-substituted hydrophobic amino acids, including alanine, leucine,isoleucine, valine, norleucine, (S)-2-aminobutyric acid,(S)-cyclohexylalanine or other simple alpha-amino acids substituted byan aliphatic side chain from C₁-C₁₀ carbons including branched, cyclicand straight chain alkyl, alkenyl or alkynyl substitutions; b)substitution of aromatic-substituted hydrophobic amino acids, includingphenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine,1-naphthylalanine, 2-naphthylalanine, 2-benzothienylalanine,3-benzothienylalanine, histidine, including amino, alkylamino,dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) oralkoxy (from C₁-C₄)-substituted forms of the above-listed aromatic aminoacids, illustrative examples of which are: 2-, 3- or4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-, 3′-, or4′-methyl-, 2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine; c)substitution of amino acids containing basic side chains, includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, including alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀branched, linear, or cyclic) derivatives of the previous amino acids,whether the substituent is on the heteroatoms (such as the alphanitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon,in the pro-R position for example. Compounds that serve as illustrativeexamples include: N-epsilon-isopropyl-lysine,3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine,N,N-gamma, gamma′-diethyl-homoarginine. Included also are compounds suchas alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl groupoccupies the pro-R position of the alpha-carbon. Also included are theamides formed from alkyl, aromatic, heteroaromatic (where theheteroaromatic group has one or more nitrogens, oxygens or sulfur atomssingly or in combination), carboxylic acids or any of the manywell-known activated derivatives such as acid chlorides, active esters,active azolides and related derivatives, and lysine, ornithine, or2,3-diaminopropionic acid; d) substitution of acidic amino acids,including aspartic acid, glutamic acid, homoglutamic acid, tyrosine,alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of2,4-diaminopriopionic acid, ornithine or lysine andtetrazole-substituted alkyl amino acids; e) substitution of side chainamide residues, including asparagine, glutamine, and alkyl or aromaticsubstituted derivatives of asparagine or glutamine; and f) substitutionof hydroxyl-containing amino acids, including serine, threonine,homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substitutedderivatives of serine or threonine.

In some cases, a TIMP polypeptide includes one or more naturallyoccurring non-genetically encoded L-amino acids, synthetic L-aminoacids, or D-enantiomers of an amino acid. For example, a TIMPpolypeptide can include only D-amino acids. For example, a TIMPpolypeptide can include one or more of the following residues:hydroxyproline, β-alanine, o-aminobenzoic acid, m-aminobenzoic acid,p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3-diaminopropionicacid, α-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine,citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine,phenylglycine, cyclohexylalanine, norleucine, naphthylalanine,pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine,2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine,penicillamine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,β-2-thienylaIanine, methionine sulfoxide, homoarginine, N-acetyl lysine,2,4-diamino butyric acid, rho-aminophenylalanine, N-methylvaline,homocysteine, homoserine, ε-amino hexanoic acid, ω-aminohexanoic acid,ω-aminoheptanoic acid, ω-aminooctanoic acid, ω-aminodecanoic acid,ω-aminotetradecanoic acid, cyclohexylalanine, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, δ-amino valeric acid, and 2,3-diaminobutyricacid.

A cysteine residue or a cysteine analog can be introduced into a TIMPpolypeptide to provide for linkage to another peptide via a disulfidelinkage or to provide for cyclization of the TIMP polypeptide. a TIMPpolypeptide can be cyclized. One or more cysteines or cysteine analogscan be introduced into a TIMP polypeptide, where the introduced cysteineor cysteine analog can form a disulfide bond with a second introducedcysteine or cysteine analog. Other means of cyclization includeintroduction of an oxime linker or a lanthionine linker; see, e.g., U.S.Pat. No. 8,044,175. Any combination of amino acids (or non-amino acidmoieties) that can form a cyclizing bond can be used and/or introduced.A cyclizing bond can be generated with any combination of amino acids(or with an amino acid and —(CH2)_(n)—CO— or —(CH2)_(n)—C₆H₄—CO—) withfunctional groups which allow for the introduction of a bridge. Someexamples are disulfides, disulfide mimetics such as the —(CH2)_(n)—carba bridge, thioacetal, thioether bridges (cystathionine orlanthionine) and bridges containing esters and ethers. In theseexamples, n can be any integer, but is frequently less than ten.

Other modifications include, for example, an N-alkyl (or aryl)substitution (ψ[CONR]), or backbone crosslinking to construct lactamsand other cyclic structures. Other derivatives include C-terminalhydroxymethyl derivatives, o-modified derivatives (e.g., C-terminalhydroxymethyl benzyl ether), N-terminally modified derivatives includingsubstituted amides such as alkylamides and hydrazides.

Modifications may be present that provide for improvements in one ormore physical properties of the TIMP polypeptide. Improvements ofphysical properties include, for example, modulating immunogenicity;methods of increasing water solubility, bioavailability, serumhalf-life, and/or therapeutic half-life; and/or modulating biologicalactivity. Examples of such modifications include, but are not limitedto: pegylation, glycosylation (N- and O-linked); polysialylation;albumin fusion molecules comprising serum albumin (e.g., human serumalbumin (HSA), cyno serum albumin, or bovine serum albumin (BSA));albumin binding through, for example a conjugated fatty acid chain(acylation); and Fc-fusion proteins.

Pegylation:

The clinical effectiveness of protein therapeutics may be limited byshort plasma half-life and susceptibility to protease degradation.Studies of various therapeutic proteins (e.g., filgrastim) have shownthat such difficulties may be overcome by various modifications,including conjugating or linking the polypeptide sequence to any of avariety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes. This is frequently effectedby a linking moiety covalently bound to both the protein and thenonproteinaceous polymer, e.g., a PEG. Such PEG-conjugated biomoleculeshave been shown to possess clinically useful properties, includingbetter physical and thermal stability, protection against susceptibilityto enzymatic degradation, increased solubility, longer in vivocirculating half-life and decreased clearance, reduced immunogenicityand antigenicity, and reduced toxicity. In addition to the beneficialeffects of pegylation on pharmacokinetic parameters, pegylation itselfmay enhance activity. PEGs suitable for conjugation to a polypeptidesequence are generally soluble in water at room temperature, and havethe general formula R(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or aprotective group such as an alkyl or an alkanol group, and where n is aninteger from 1 to 1000. When R is a protective group, it generally hasfrom 1 to 8 carbons. The PEG conjugated to the polypeptide sequence canbe linear or branched. Branched PEG derivatives, “star-PEGs” andmulti-armed PEGs are contemplated by the present disclosure. A molecularweight of the PEG used in the present disclosure is not restricted toany particular range, and examples are set forth elsewhere herein; byway of example, certain embodiments have molecular weights between 5 kDaand 20 kDa, while other embodiments have molecular weights between 4 kDaand 10 kDa. Pegylated TIMP polypeptides may be conjugates wherein thePEGs have different n values, and thus the various different PEGs arepresent in specific ratios. For example, some compositions comprise amixture of conjugates where n=1, 2, 3 and 4. In some compositions, thepercentage of conjugates where n=1 is 18-25%, the percentage ofconjugates where n=2 is 50-66%, the percentage of conjugates where n=3is 12-16%, and the percentage of conjugates where n=4 is up to 5%. Suchcompositions can be produced by any convenient reaction conditions andpurification. Pegylation most frequently occurs at the alpha amino groupat the N-terminus of the polypeptide, the epsilon amino group on theside chain of lysine residues, and the imidazole group on the side chainof histidine residues. Since most recombinant polypeptides possess asingle alpha and a number of epsilon amino and imidazole groups,numerous positional isomers can be generated depending on the linkerchemistry. General pegylation strategies, such as those known in theart, can be applied herein. PEG may be bound to a polypeptide of thepresent disclosure via a terminal reactive group (a “spacer”) whichmediates a bond between the free amino or carboxyl groups of one or moreof the polypeptide sequences and polyethylene glycol. The PEG having thespacer which may be bound to the free amino group includesN-hydroxysuccinylimide polyethylene glycol which may be prepared byactivating succinic acid ester of polyethylene glycol withN-hydroxysuccinylimide. Another activated polyethylene glycol which maybe bound to a free amino group is2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, which may beprepared by reacting polyethylene glycol monomethyl ether with cyanuricchloride. The activated polyethylene glycol which is bound to the freecarboxyl group includes polyoxyethylenediamine. Conjugation of one ormore of the polypeptide sequences to PEG having a spacer may be carriedout by various conventional methods. For example, the conjugationreaction can be carried out in solution at a pH of from 5 to 10, attemperature from 4° C. to room temperature, for 30 minutes to 20 hours,utilizing a molar ratio of reagent to protein of from 4:1 to 30:1.Reaction conditions may be selected to direct the reaction towardsproducing predominantly a desired degree of substitution. In general,low temperature, low pH (e.g., pH=5), and short reaction time tend todecrease the number of PEGs attached, whereas high temperature, neutralto high pH (e.g., pH≥7), and longer reaction time tend to increase thenumber of PEGs attached. Various means known in the art may be used toterminate the reaction. In some embodiments the reaction is terminatedby acidifying the reaction mixture and freezing at, e.g., −20° C.Pegylation of various molecules is discussed in, for example, U.S. Pat.Nos. 5,252,714; 5,643,575; 5,919,455; 5,932,462; and 5,985,263. Thepresent disclosure also contemplates the use of PEG mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half-life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the peptide or protein drug ofinterest. This obviates the need for an additional conjugation stepduring the manufacturing process. Moreover, established molecularbiology techniques enable control of the side chain composition of thepolypeptide chains, allowing optimization of immunogenicity andmanufacturing properties.

Glycosylation:

For purposes of the present disclosure, “glycosylation” is meant tobroadly refer to the enzymatic process that attaches glycans toproteins, lipids or other organic molecules. The use of the term“glycosylation” in conjunction with the present disclosure is generallyintended to mean adding or deleting one or more carbohydrate moieties(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that may or may not be present in the nativesequence. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins involving a change in the natureand proportions of the various carbohydrate moieties present.Glycosylation can dramatically affect the physical properties (e.g.,solubility) of polypeptides such as TIMP polypeptides and can also beimportant in protein stability, secretion, and subcellular localization.Glycosylated polypeptides may also exhibit enhanced stability or mayimprove one or more pharmacokinetic properties, such as half-life. Inaddition, solubility improvements can, for example, enable thegeneration of formulations more suitable for pharmaceuticaladministration than formulations comprising the non-glycosylatedpolypeptide. Addition of glycosylation sites can be accomplished byaltering the amino acid sequence. The alteration to the polypeptide maybe made, for example, by the addition of, or substitution by, one ormore serine or threonine residues (for O-linked glycosylation sites) orasparagine residues (for N-linked glycosylation sites). The structuresof N-linked and O-linked oligosaccharides and the sugar residues foundin each type may be different. One type of sugar that is commonly foundon both is N-acetylneuraminic acid (hereafter referred to as sialicacid). Sialic acid is usually the terminal residue of both N-linked andO-linked oligosaccharides and, by virtue of its negative charge, mayconfer acidic properties to the glycoprotein. A particular embodiment ofthe present disclosure comprises the generation and use ofN-glycosylation variants. The polypeptide sequences of the presentdisclosure may optionally be altered through changes at the nucleic acidlevel, particularly by mutating the nucleic acid encoding thepolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids. Another means of increasingthe number of carbohydrate moieties on the polypeptide is by chemical orenzymatic coupling of glycosides to the polypeptide. Removal ofcarbohydrates may be accomplished chemically or enzymatically, or bysubstitution of codons encoding amino acid residues that areglycosylated. Chemical deglycosylation techniques are known, andenzymatic cleavage of carbohydrate moieties on polypeptides can beachieved by the use of a variety of endo- and exo-glycosidases.Dihydrofolate reductase (DHFR)-deficient Chinese Hamster Ovary (CHO)cells are a commonly used host cell for the production of recombinantglycoproteins. These cells do not express the enzyme beta-galactosidealpha-2,6-sialyltransferase and therefore do not add sialic acid in thealpha-2,6 linkage to N-linked oligosaccharides of glycoproteins producedin these cells.

In some embodiments, the polypeptides are non-naturally glycosylated. Bynon-naturally glycosylated is meant that the polypeptide has aglycosylation pattern, if present, which is not the same as theglycosylation pattern found in the corresponding naturally occurringprotein. For example, a human TIMP2 employed in methods of the inventionof this particular embodiment is characterized by having a glycosylationpattern, if glycosylated at all, that differs from that of naturallyoccurring human TIMP2. Thus, the non-naturally glycosylated TIMPpolypeptides of this embodiment include non-glycosylated TIMPpolypeptides, i.e. proteins having no covalently bound glycosyl groups.

Polysialylation:

The present disclosure also contemplates the use of polysialylation, theconjugation of polypeptides to the naturally occurring, biodegradableα-(2→8) linked polysialic acid (“PSA”) in order to improve thepolypeptides' stability and in vivo pharmacokinetics. PSA is abiodegradable, non-toxic natural polymer that is highly hydrophilic,giving it a high apparent molecular weight in the blood which increasesits serum half-life. In addition, polysialylation of a range of peptideand protein therapeutics has led to markedly reduced proteolysis,retention of in vivo activity, and reduction in immunogenicity andantigenicity (see, e.g., G. Gregoriadis et al., Int. J. Pharmaceutics300(1-2):125-30). As with modifications with other conjugates (e.g.,PEG), various techniques for site-specific polysialylation are available(see, e.g., T. Lindhout et al., (2011) PNAS 108(18)7397-7402).

Albumin Fusion:

Additional suitable components and molecules for conjugation includealbumins such as human serum albumin (HSA), cyno serum albumin, andbovine serum albumin (BSA). Mature HSA, a 585 amino acid polypeptide(˜67 kDa) having a serum half-life of ˜20 days, is primarily responsiblefor the maintenance of colloidal osmotic blood pressure, blood pH, andtransport and distribution of numerous endogenous and exogenous ligands.The protein has three structurally homologous domains (domains I, II andIII), is almost entirely in the alpha-helical conformation, and ishighly stabilized by 17 disulphide bridges. The three primary drugbinding regions of albumin are located on each of the three domainswithin sub-domains IB, IIA and IIIA. Albumin synthesis takes place inthe liver, which produces the short-lived, primary productpreproalbumin. Thus, the full-length HSA has a signal peptide of 18amino acids (MKWVTFISLLFLFSSAYS; SEQ ID NO:5) followed by a pro-domainof 6 amino acids (RGVFRR; SEQ ID NO:6); this 24 amino acid residuepeptide may be referred to as the pre-pro domain. HSA can be expressedand secreted using its endogenous signal peptide as a pre-pro-domain.Alternatively, HSA can be expressed and secreted using a IgK signalpeptide fused to a mature construct. Preproalbumin is rapidlyco-translationally cleaved in the endoplasmic reticulum lumen at itsamino terminus to produce the stable, 609-amino acid precursorpolypeptide, proalbumin. Proalbumin then passes to the Golgi apparatus,where it is converted to the 585 amino acid mature albumin by afurin-dependent amino-terminal cleavage. The primary amino acidsequences, structure, and function of albumins are highly conservedacross species, as are the processes of albumin synthesis and secretion.Albumin serum proteins comparable to HSA are found in, for example,cynomolgus monkeys, cows, dogs, rabbits and rats. Of the non-humanspecies, bovine serum albumin (BSA) is the most structurally similar toHSA (see, e.g., Kosa et al., November 2007 J Pharm Sci. 96(11):3117-24).The present disclosure contemplates the use of albumin from non-humanspecies, including, but not limited to, those set forth above, in, forexample, the drug development process. According to the presentdisclosure, albumin may be conjugated to a drug molecule (e.g., apolypeptide described herein) at the carboxyl terminus, the aminoterminus, both the carboxyl and amino termini, and internally (see,e.g., U.S. Pat. Nos. 5,876,969 and 7,056,701). In the HSA-TIMPconjugates contemplated by the present disclosure, various forms ofalbumin may be used, such as albumin secretion pre-sequences andvariants thereof, fragments and variants thereof, and HSA variants. Suchforms generally possess one or more desired albumin activities. Inadditional embodiments, the present disclosure involves fusion proteinscomprising a polypeptide drug molecule fused directly or indirectly toalbumin, an albumin fragment, and albumin variant, etc., wherein thefusion protein has a higher plasma stability than the unfused drugmolecule and/or the fusion protein retains the therapeutic activity ofthe unfused drug molecule. In some embodiments, the indirect fusion iseffected by a linker, such as a peptide linker or modified versionthereof. Intracellular cleavage may be carried out enzymatically by, forexample, furin or caspase. Cells express a low level of these endogenousenzymes, which are capable of cleaving a portion of the fusion moleculesintracellularly; thus, some of the polypeptides are secreted from thecell without being conjugated to HSA, while some of the polypeptides aresecreted in the form of fusion molecules that comprise HSA. Embodimentsof the present disclosure contemplate the use of various furin fusionconstructs. For example, constructs may be designed that comprise thesequence RGRR (SEQ ID NO:19), RKRKKR (SEQ ID NO:20), RKKR (SEQ IDNO:21), or RRRKKR (SEQ ID NO:22). The present disclosure alsocontemplates extracellular cleavage (i.e., ex-vivo cleavage) whereby thefusion molecules are secreted from the cell, subjected to purification,and then cleaved. It is understood that the excision may dissociate theentire HSA-linker complex from the mature TIMP polypeptide, or less thatthe entire HSA-linker complex. As alluded to above, fusion of albumin toone or more polypeptides of the present disclosure can, for example, beachieved by genetic manipulation, such that the nucleic acid coding forHSA, or a fragment thereof, is joined to the nucleic acid coding for theone or more polypeptide sequences. Thereafter, a suitable host can betransformed or transfected with the fused nucleotide sequences in theform of, for example, a suitable plasmid, so as to express a fusionpolypeptide. The expression may be effected in vitro from, for example,prokaryotic or eukaryotic cells, or in vivo from, for example, atransgenic organism. In some embodiments of the present disclosure, theexpression of the fusion protein is performed in mammalian cell lines,for example, CHO cell lines. Transformation is used broadly herein torefer to the genetic alteration of a cell resulting from the directuptake through the cell membrane, incorporation and expression ofexogenous genetic material (exogenous nucleic acid). Transformationoccurs naturally in some species of bacteria, but it can also beeffected by artificial means in other cells. Furthermore, albumin itselfmay be modified to extend its circulating half-life. Fusion of themodified albumin to a TIMP polypeptide can be attained by the geneticmanipulation techniques described above or by chemical conjugation; theresulting fusion molecule has a half-life that exceeds that of fusionswith non-modified albumin. TIMP2-albumin fusion proteins of interestinclude those described in U.S. Pat. No. 7,163,805, the disclosure ofwhich is herein incorporated by reference.

Several albumin—binding strategies have been developed as alternativesto direct fusion, including albumin binding through a conjugated fattyacid chain (acylation). Because serum albumin is a transport protein forfatty acids, these natural ligands with albumin—binding activity havebeen used for half-life extension of small protein therapeutics. Forexample, insulin determir (LEVEMIR), an approved product for diabetes,comprises a myristyl chain conjugated to a genetically-modified insulin,resulting in a long-acting insulin analog. The present disclosure alsocontemplates fusion proteins which comprise an albumin binding domain(ABD) polypeptide sequence and the sequence of one or more of thepolypeptides described herein. Any ABD polypeptide sequence described inthe literature can be a component of the fusion proteins. The componentsof the fusion proteins can be optionally covalently bonded through alinker, such as those linkers described herein. In some of theembodiments of the present disclosure, the fusion proteins comprise theABD polypeptide sequence as an N-terminal moiety and the polypeptidesdescribed herein as a C-terminal moiety. The present disclosure alsocontemplates fusion proteins comprising a fragment of an albumin bindingpolypeptide, which fragment substantially retains albumin binding; or amultimer of albumin binding polypeptides or their fragments comprisingat least two albumin binding polypeptides or their fragments as monomerunits.

Conjugation with Other Molecules:

Additional suitable components and molecules for conjugation include,for example, thyroglobulin; tetanus toxoid; Diphtheria toxoid; polyaminoacids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides ofrotaviruses; influenza virus hemaglutinin, influenza virusnucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B viruscore protein and surface antigen; or any combination of the foregoing.Thus, the present disclosure contemplates conjugation of one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another polypeptide (e.g., a polypeptidehaving an amino acid sequence heterologous to the subject polypeptide),or a carrier molecule. Thus, an exemplary polypeptide sequence can beprovided as a conjugate with another component or molecule. A conjugatemodification may result in a polypeptide sequence that retains activitywith an additional or complementary function or activity derived fromthe second molecule. For example, a polypeptide sequence may beconjugated to a molecule, e.g., to facilitate solubility, storage, invivo or shelf half-life or stability, reduction in immunogenicity,delayed or controlled release in vivo, etc. Other functions oractivities include a conjugate that reduces toxicity relative to anunconjugated polypeptide sequence, a conjugate that targets a type ofcell or organ more efficiently than an unconjugated polypeptidesequence, or a drug to further counter the causes or effects associatedwith a disease, disorder or condition as set forth herein (e.g.,cancer). A TIMP polypeptide may also be conjugated to large, slowlymetabolized macromolecules such as proteins; polysaccharides, such assepharose, agarose, cellulose, or cellulose beads; polymeric amino acidssuch as polyglutamic acid, or polylysine; amino acid copolymers;inactivated virus particles; inactivated bacterial toxins such as toxoidfrom diphtheria, tetanus, cholera, or leukotoxin molecules; inactivatedbacteria; and dendritic cells. Such conjugated forms, if desired, can beused to produce antibodies against a polypeptide of the presentdisclosure. Additional candidate components and molecules forconjugation include those suitable for isolation or purification.Particular non-limiting examples include binding molecules, such asbiotin (biotin-avidin specific binding pair), an antibody, a receptor, aligand, a lectin, or molecules that comprise a solid support, including,for example, plastic or polystyrene beads, plates or beads, magneticbeads, test strips, and membranes. Purification methods such as cationexchange chromatography may be used to separate conjugates by chargedifference, which effectively separates conjugates into their variousmolecular weights. For example, the cation exchange column can be loadedand then washed with ˜20 mM sodium acetate, pH ˜4, and then eluted witha linear (0 M to 0.5 M) NaCl gradient buffered at a pH from about 3 to5.5, e.g., at pH ˜4.5. The content of the fractions obtained by cationexchange chromatography may be identified by molecular weight usingconventional methods, for example, mass spectroscopy, SDS-PAGE, or otherknown methods for separating molecular entities by molecular weight.

Fc-Fusion Molecules:

In certain embodiments, the amino- or carboxyl-terminus of a polypeptidesequence of the present disclosure can be fused with an immunoglobulinFc region (e.g., human Fc) to form a fusion conjugate (or fusionmolecule). Fc fusion conjugates have been shown to increase the systemichalf-life of biopharmaceuticals, and thus the biopharmaceutical productmay require less frequent administration. Fc binds to the neonatal Fcreceptor (FcRn) in endothelial cells that line the blood vessels, and,upon binding, the Fc fusion molecule is protected from degradation andre-released into the circulation, keeping the molecule in circulationlonger. This Fc binding is believed to be the mechanism by whichendogenous IgG retains its long plasma half-life. More recent Fc-fusiontechnology links a single copy of a biopharmaceutical to the Fc regionof an antibody to optimize the pharmacokinetic and pharmacodynamicproperties of the biopharmaceutical as compared to traditional Fc-fusionconjugates.

Other Modifications:

The present disclosure contemplates the use of other modifications,currently known or developed in the future, of TIMP polypeptides toimprove one or more properties. One such method for prolonging thecirculation half-life, increasing the stability, reducing the clearance,or altering the immunogenicity or allergenicity of a polypeptide of thepresent disclosure involves modification of the polypeptide sequences byhesylation, which utilizes hydroxyethyl starch derivatives linked toother molecules in order to modify the polypeptide sequences'characteristics.

Linkers:

Linkers and their use have been described above. Any of the foregoingcomponents and molecules used to modify the polypeptide sequences of thepresent disclosure may optionally be conjugated via a linker. Suitablelinkers include “flexible linkers” which are generally of sufficientlength to permit some movement between the modified polypeptidesequences and the linked components and molecules. The linker moleculesare generally about 6-50 atoms long. The linker molecules may also be,for example, aryl acetylene, ethylene glycol oligomers containing 2-10monomer units, diamines, diacids, amino acids, or combinations thereof.Suitable linkers can be readily selected and can be of any suitablelength, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10,10-20, 20-30, 30-50 or more than 50 amino acids. Exemplary flexiblelinkers include glycine polymers (G)_(n), glycine-serine polymers (forexample, (GS)_(n), GSGGS_(n) (SEQ ID NO:7), GGGS_(n)(SEQ ID NO:8),(G_(m)S_(o))_(n), (G_(m)S_(o)G_(m))_(n), (G_(m)S_(o)G_(m)S_(o)G_(m))_(n)(SEQ ID NO:9), (GSGGS_(m))_(n) (SEQ ID NO:10), (GSGS_(m)G)_(n) (SEQ IDNO:11) and (GGGS_(m))_(n) (SEQ ID NO:12), and combinations thereof,where m, and o are each independently selected from an integer of atleast one), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers. Glycine and glycine-serine polymers are relativelyunstructured, and therefore may serve as a neutral tether betweencomponents. Exemplary flexible linkers include, but are not limited toGGSG (SEQ ID NO:13), GGSGG (SEQ ID NO:14), GSGSG (SEQ ID NO:15), GSGGG(SEQ ID NO:16), GGGSG (SEQ ID NO:17), and GSSSG (SEQ ID NO:18).

In some instances, systemic TIMP polypeptide levels is increased byadministering a nucleic acid coding sequence to the subject underconditions sufficient for the coding sequence to be expressed in thesubject. Depending on the desired TIMP polypeptide, the nucleic acidcoding sequence may vary. Nucleic acids of interest include thoseencoding the TIMP polypeptides provided above. Specific nucleic acids ofinterest include, but are not limited to: Human TIMP2 (NCBI ReferenceSequence: NM_003255.4); Human TIMP1 (NCBI Reference Sequence:NM_003254.2); Human TIMP3 (NCBI Reference Sequence: NM_000362.4) andHuman TIMP4 (NCBI Reference Sequence: NM_003256.3).

By nucleic acid composition is meant a composition comprising a sequenceof DNA having an open reading frame that encodes a TIMP polypeptide ofinterest, i.e., a TIMP coding sequence, and is capable, underappropriate conditions, of being expressed as a TIMP polypeptide. Alsoencompassed in this term are nucleic acids that are homologous,substantially similar or identical to the specific nucleic acidsdescribed above. In addition to the above described specific nucleicacid compositions, also of interest are homologues of the abovesequences. In certain embodiments, sequence similarity betweenhomologues is 20% or higher, such as 25% or higher, and including 30%,35%, 40%, 50%, 60%, 70% or higher, including 75%, 80%, 85%, 90% and 95%or higher. Sequence similarity is calculated based on a referencesequence, which may be a subset of a larger sequence, such as aconserved motif, coding region, flanking region, etc. A referencesequence may be 18 nt long or longer, such as 30 nt long, and may extendto the complete sequence that is being compared. Algorithms for sequenceanalysis are known in the art, such as BLAST, described in Altschul etal. (1990), J. Mol. Biol. 215:403-10 (using default settings, i.e.parameters w=4 and T=17). Of particular interest in certain embodimentsare nucleic acids of substantially the same length as specific humanTIMP1 to TIMP4 nucleic acids mentioned above, where by substantially thesame length is meant that any difference in length does not exceed about20 number %, usually does not exceed about 10 number and more usuallydoes not exceed about 5 number %; and have sequence identity to any ofthese sequences of at 90% or greater, such as 95% or greater andincluding 99% or greater over the entire length of the nucleic acid. Insome embodiments, the nucleic acids have a sequence that issubstantially similar or identical to the above specific sequences. Bysubstantially similar is meant that sequence identity is 60% or greater,such as 75% or greater and including 80, 85, 90, or even 95% or greater.Nucleic acids of interest also include nucleic acids that encode theproteins encoded by the above described nucleic acids, but differ insequence from the above described nucleic acids due to the degeneracy ofthe genetic code.

Nucleic acids as described herein may be present in a vector. Variousvectors (e.g., viral vectors, bacterial vectors, or vectors capable ofreplication in eukaryotic and prokaryotic hosts) can be used inaccordance with the present invention. Numerous vectors which canreplicate in eukaryotic and prokaryotic hosts are known in the art andare commercially available. In some instances, such vectors used inaccordance with the invention are composed of a bacterial origin ofreplication and a eukaryotic promoter operably linked to a DNA ofinterest.

Viral vectors used in accordance with the invention may be composed of aviral particle derived from a naturally-occurring virus which has beengenetically altered to render the virus replication-defective and toexpress a recombinant gene of interest in accordance with the invention.Once the virus delivers its genetic material to a cell, it does notgenerate additional infectious virus but does introduce exogenousrecombinant genes into the cell, preferably into the genome of the cell.Numerous viral vectors are well known in the art, including, forexample, retrovirus, adenovirus, adeno-associated virus, herpes simplexvirus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus vectors.

The DNA of interest may be administered using a non-viral vector, forexample, as a DNA- or RNA-liposome complex formulation. Such complexescomprise a mixture of lipids which bind to genetic material (DNA orRNA), providing a hydrophobic coat which allows the genetic material tobe delivered into cells. Liposomes which can be used in accordance withthe invention include DOPE (dioleyl phosphatidyl ethanol amine), CUDMEDA(N-(5-cholestrum-3-.beta.-ol 3-urethanyl)-N′,N′-dimethylethylenediamine). When the DNA of interest is introduced using a liposome, insome instances one first determines in vitro the optimal values for theDNA: lipid ratios and the absolute concentrations of DNA and lipid as afunction of cell death and transformation efficiency for the particulartype of cell to be transformed. These values can then be used in orextrapolated for use in in vivo transformation. The in vitrodeterminations of these values can be readily carried out usingtechniques which are well known in the art.

Other non-viral vectors may also be used in accordance with the presentinvention. These include chemical formulations of DNA or RNA coupled toa carrier molecule (e.g., an antibody or a receptor ligand) whichfacilitates delivery to host cells for the purpose of altering thebiological properties of the host cells. By the term “chemicalformulations” is meant modifications of nucleic acids to allow couplingof the nucleic acid compounds to a carrier molecule such as a protein orlipid, or derivative thereof. Exemplary protein carrier moleculesinclude antibodies specific to the cells of a targeted secretory glandor receptor ligands, i.e., molecules capable of interacting withreceptors associated with a cell of a targeted secretory gland.

DNA constructs may include a promoter to facilitate expression of theDNA of interest within a target cell, such as a strong, eukaryoticpromoter. Exemplary eukaryotic promoters include promoters fromcytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Rous sarcomavirus (RSV), and adenovirus. More specifically, exemplary promotersinclude the promoter from the immediate early gene of human CMV (Boshartet al., Cell 41:521-530, 1985) and the promoter from the long terminalrepeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci. USA79:6777-6781, 1982).

Instead of administration of a TIMP polypeptide, e.g., as describedabove, the level of systemic TIMP active agent in the subject may beenhanced by stimulating endogenous production and/or release of a TIMPpolypeptide in vivo.

Also of interest are potentiators of TIMP activity. By TIMP potentiatoris meant an agent or combination of agents that work to increase thedesirable TIMP activity of endogenous TIMP polypeptides present in thesubject being treated. The magnitude of the increase may vary, where insome instances the magnitude of the increase is 2-fold or greater, suchas 5-fold or greater, including 10-fold or greater, e.g., 15-fold orgreater, 20-fold or greater, 25-fold or greater (as compared to asuitable control). TIMP potentiators of interest may work through avariety of different mechanisms, e.g., by enhancing the bindinginteraction between a TIMP polypeptide and a desired target; byincreasing the bioavailability of the endogenous pool, e.g., bysequestering undesirable competitive binding targets, etc.

In yet other embodiments, the agent is a small molecule agent thatexhibits the desired TIMP activity. Naturally occurring or syntheticsmall molecule compounds of interest include numerous chemical classes,such as organic molecules, e.g., small organic compounds having amolecular weight of more than 50 and less than about 2,500 Daltons.Candidate agents comprise functional groups for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, preferably atleast two of the functional chemical groups. The candidate agents mayinclude cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Such molecules may be identified, among other ways, by employing thescreening protocols described below.

In practicing methods of the invention, the active agent(s) may beadministered to the adult mammal using any convenient administrationprotocol capable of resulting in the desired activity. Thus, the agentcan be incorporated into a variety of formulations, e.g.,pharmaceutically acceptable vehicles, for therapeutic administration.More particularly, the agents of the present invention can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments (e.g., skin creams),solutions, suppositories, injections, inhalants and aerosols. As such,administration of the agents can be achieved in various ways, includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal,transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, it may be introduced into tissues or host cells by any numberof routes, including viral infection, microinjection, or fusion ofvesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al., Anal Biochem. (1992)205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al., Nature(1992) 356:152-154), where gold microprojectiles are coated with theDNA, then bombarded into skin cells. For nucleic acid therapeuticagents, a number of different delivery vehicles find use, includingviral and non-viral vector systems, as are known in the art.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Preferred dosages for a given compound arereadily determinable by those of skill in the art by a variety of means.

In those embodiments where an effective amount of an active agent isadministered to the adult mammal, the amount or dosage is effective whenadministered for a suitable period of time, such as one week or longer,including two weeks or longer, such as 3 weeks or longer, 4 weeks orlonger, 8 weeks or longer, etc., so as to evidence a reduction in theimpairment, e.g., cognition decline and/or cognitive improvement in theadult mammal. For example, an effective dose is the dose that, whenadministered for a suitable period of time, such as at least about oneweek, and maybe about two weeks, or more, up to a period of about 3weeks, 4 weeks, 8 weeks, or longer, will slow e.g., by about 20% ormore, e.g., by 30% or more, by 40% or more, or by 50% or more, in someinstances by 60% or more, by 70% or more, by 80% or more, or by 90% ormore, e.g., will halt, cognitive decline in a patient suffering fromnatural aging or an aging-associated disorder. In some instances, aneffective amount or dose of active agent will not only slow or halt theprogression of the disease condition but will also induce the reversalof the condition, i.e., will cause an improvement in cognitive ability.For example, in some instances, an effective amount is the amount thatwhen administered for a suitable period of time, usually at least aboutone week, and maybe about two weeks, or more, up to a period of about 3weeks, 4 weeks, 8 weeks, or longer will improve the cognitive abilitiesof an individual suffering from an aging-associated cognitive impairmentby, for example 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, in someinstances 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more relative tocognition prior to administration of the blood product.

Where desired, effectiveness of treatment may be assessed using anyconvenient protocol. Cognition tests and IQ test for measuring cognitiveability, e.g., attention and concentration, the ability to learn complextasks and concepts, memory, information processing, visuospatialfunction, the ability to produce and understanding language, the abilityto solve problems and make decisions, and the ability to performexecutive functions, are well known in the art, any of which may be usedto measure the cognitive ability of the individual before and/or duringand after treatment with the subject blood product, e.g., to confirmthat an effective amount has been administered. These include, forexample, the General Practitioner Assessment of Cognition (GPCOG) test,the Memory Impairment Screen, the Mini Mental State Examination (MMSE),the California Verbal Learning Test, Second Edition, Short Form, formemory, the Delis-Kaplan Executive Functioning System test, theAlzheimer's Disease Assessment Scale (ADAS-Cog), the PsychogeriatricAssessment Scale (PAS) and the like. Progression of functional brainimprovements may be detected by brain imaging techniques, such asMagnetic Resonance Imaging (MRI) or Positron Emission Tomography (PET)and the like. A wide range of additional functional assessments may beapplied to monitor activities of daily living, executive functions,mobility, etc. In some embodiments, the method comprises the step ofmeasuring cognitive ability, and detecting a decreased rate of cognitivedecline, a stabilization of cognitive ability, and/or an increase incognitive ability after administration of the blood product as comparedto the cognitive ability of the individual before the blood product wasadministered. Such measurements may be made a week or more afteradministration of the blood product, e.g., 1 week, 2 weeks, 3 weeks, ormore, for instance, 4 weeks, 6 weeks, or 8 weeks or more, e.g., 3months, 4 months, 5 months, or 6 months or more.

Biochemically, by an “effective amount” or “effective dose” of activeagent is meant an amount of active agent that will inhibit, antagonize,decrease, reduce, or suppress by about 20% or more, e.g., by 30% ormore, by 40% or more, or by 50% or more, in some instances by 60% ormore, by 70% or more, by 80% or more, or by 90% or more, in some casesby about 100%, i.e., to negligible amounts, and in some instancesreverse, the reduction in synaptic plasticity and loss of synapses thatoccurs during the natural aging process or during the progression of anaging-associated disorder. In other words, cells present in adultmammals treated in accordance with methods of the invention will becomemore responsive to cues, e.g., activity cues, which promote theformation and maintenance of synapses.

Performance of methods of the invention, e.g., as described above, maymanifest as improvements in observed synaptic plasticity, both in vitroand in vivo as an induction of long term potentiation. For example, theinduction of LTP in neural circuits may be observed in awakeindividuals, e.g., by performing non-invasive stimulation techniques onawake individuals to induce LTP-like long-lasting changes in localizedneural activity (Cooke S F, Bliss T V (2006) Plasticity in the humancentral nervous system. Brain. 129(Pt 7):1659-73); mapping plasticityand increased neural circuit activity in individuals, e.g., by usingpositron emission tomography, functional magnetic resonance imaging,and/or transcranial magnetic stimulation (Cramer and Bastings, “Mappingclinically relevant plasticity after stroke,” Neuropharmacology(2000)39:842-51); and by detecting neural plasticity following learning,i.e., improvements in memory, e.g., by assaying retrieval-related brainactivity (Buchmann et al., “Prion protein M129V polymorphism affectsretrieval-related brain activity,” Neuropsychologia. (2008) 46:2389-402)or, e.g., by imaging brain tissue by functional magnetic resonanceimaging (fMRI) following repetition priming with familiar and unfamiliarobjects (Soldan et al., “Global familiarity of visual stimuli affectsrepetition-related neural plasticity but not repetition priming,”Neuroimage. (2008) 39:515-26; Soldan et al., “Aging does not affectbrain patterns of repetition effects associated with perceptual primingof novel objects,” J. Cogn. Neurosci. (2008) 20:1762-76). In someembodiments, the method includes the step of measuring synapticplasticity, and detecting a decreased rate of loss of synapticplasticity, a stabilization of synaptic plasticity, and/or an increasein synaptic plasticity after administration of the blood product ascompared to the synaptic plasticity of the individual before the bloodproduct was administered. Such measurements may be made a week or moreafter administration of the blood product, e.g., 1 week, 2 weeks, 3weeks, or more, for instance, 4 weeks, 6 weeks, or 8 weeks or more,e.g., 3 months, 4 months, 5 months, or 6 months or more.

In some instances, the methods result in a change in expression levelsof one or more genes in one or more tissues of the host, e.g., ascompared to a suitable control (such as described in the Experimentalsection, below). The change in expression level of a given gene may be0.5 fold or greater, such as 1.0 fold or greater, including 1.5 fold orgreater. The tissue may vary, and in some instances is nervous systemtissue, e.g., central nervous system tissue, including brain tissue,e.g., hippocampal tissue. In some instances, the modulation ofhippocampal gene expression is manifested as enhanced hippocampalplasticity, e.g., as compared to a suitable control. In some instances,the one or more genes whose expression is modulated, e.g., enhanced, isa gene encoding a product that is a member of a plasticity relatedsignaling pathway (i.e., a synaptic plasticity regulation gene), e.g.,TIr4, Gria1, Kcnj10, Kdr, Ncam, Sdfr1, Egr1, Fos proteins, e.g., c-Fos,Drd1a, Stxbp1, Mef2c, Cntn2, Junb, Bdnf and CamK2a, etc. In someinstances, the modulation of hippocampal gene expression is manifestedas enhanced hippocampal plasticity, e.g., as compared to a suitablecontrol. In some instances, the one or more genes whose expression ismodulated, e.g., enhanced, is a gene encoding a product that is a memberof network related to synaptic plasticity and learning and memory, suchas but not limited to: RELN, NTRK3, EPHA4, etc.

In some instances, treatment results in an enhancement in the levels ofone or more proteins in one or more tissues of the host, e.g., ascompared to a suitable control (such as described in the Experimentalsection, below). The change in protein level of a given protein may be0.5 fold or greater, such as 1.0 fold or greater, including 1.5 fold orgreater, where in some instances the level may approach that of ahealthy wild-type level, e.g., within 50% or less, such as 25% or less,including 10% or less, e.g., 5% or less of the healthy wild-type level.The tissue may vary, and in some instances is nervous system tissue,e.g., central nervous system tissue, including brain tissue, e.g.,hippocampal tissue.

In some instances, the methods result in one or more structural changesin one or more tissues. The tissue may vary, and in some instances isnervous system tissue, e.g., central nervous system tissue, includingbrain tissue, e.g., hippocampal tissue. Structure changes of interestinclude an increase in dendritic spine density of mature neurons in thedentate gyrus (DG) of the hippocampus, e.g., as compared to a suitablecontrol. In some instances, the modulation of hippocampal structure ismanifested as enhanced synapse formation or function, e.g., as comparedto a suitable control. In some instances, the methods may result in anenhancement of long term potentiation, e.g., as compared to a suitablecontrol.

In some instances, practice of the methods, e.g., as described above,results in an increase in neurogenesis in the adult mammal. The increasemay be identified in a number of different ways, e.g., as describedbelow in the Experimental section. In some instances, the increase inneurogenesis manifests as an increase the amount of Dcx-positiveimmature neurons, e.g., where the increase may be 1.5-fold or greater.In some instances, the increase in neurogenesis manifests as an increasein the number of BrdU/NeuN positive cells, where the increase may be1.5-fold or greater.

In some instances, the methods result in enhancement in learning andmemory, e.g., as compared to a suitable control. Enhancement in learningand memory may be evaluated in a number of different ways, e.g., thecontextual fear conditioning, Barnes maze, and/or radial arm water maze(RAWM) paradigms described in the experimental section, below. Whenmeasured by contextual fear conditioning, treatment results in someinstances in increased freezing in contextual, but not cued, memorytesting. When measured by Barnes maze, treatment results in someinstances in enhanced learning and memory for escape hole locationduring the testing phase of the task on any day of the task. Whenmeasured by RAWM, treatment results in some instances in enhancedlearning and memory for platform location during the testing phase ofthe task. In some instances, treatment is manifested as enhancedcognitive improvement in hippocampal-dependent learning and memory,e.g., as compared to a suitable control.

In some embodiments, the methods may be performed in conjunction with anactive agent having activity suitable to treat aging-associatedcognitive impairment. For example, a number of active agents have beenshown to have some efficacy in treating the cognitive symptoms ofAlzheimer's disease (e.g., memory loss, confusion, and problems withthinking and reasoning), e.g., cholinesterase inhibitors (e.g.,Donepezil, Rivastigmine, Galantamine, Tacrine), Memantine, and VitaminE. As another example, a number of agents have been shown to have someefficacy in treating behavioral or psychiatric symptoms of Alzheimer'sDisease, e.g., citalopram (Celexa), fluoxetine (Prozac), paroxeine(Paxil), sertraline (Zoloft), trazodone (Desyrel), lorazepam (Ativan),oxazepam (Serax), aripiprazole (Abilify), clozapine (Clozaril),haloperidol (Haldol), olanzapine (Zyprexa), quetiapine (Seroquel),risperidone (Risperdal), and ziprasidone (Geodon).

In some instances, the methods are practiced in conjunction with one ormore additional non-TIMP polypeptides active agents, where such non-TIMPpolypeptide active agent exhibit a desirable anti-aging associatedcondition activity, e.g., as described above. Examples of such non-TIMPpolypeptide active agents include, but are not limited to: chemokine(C—C motif) ligand 2 (CCL2) (i.e., MCP1) and C—C motif chemokine 11(i.e., chemotactic protein or eotaxin-1) and agonists/mimetics thereof(e.g., as described in published application no. 20130040844, thedisclosure of which is herein incorporated by reference0;Granulocyte-macrophage colony-stimulating factor (GM-CSF)(i.e., colonystimulating factor 2 or CSF2); etc. In such instances, the active agentmay be any type of convenient active agent, including those types ofagents discussed above in connection with TIMP active agents, e.g.,polypeptides and mimetics/fragments thereof, small molecules, nucleicacids, potentiators, etc.

In some aspects of the subject methods, the method further comprises thestep of measuring cognition and/or synaptic plasticity after treatment,e.g., using the methods described herein or known in the art, anddetermining that the rate of cognitive decline or loss of synapticplasticity have been reduced and/or that cognitive ability or synapticplasticity have improved in the individual. In some such instances, thedetermination is made by comparing the results of the cognition orsynaptic plasticity test to the results of the test performed on thesame individual at an earlier time, e.g., 2 weeks earlier, 1 monthearlier, 2 months earlier, 3 months earlier, 6 months earlier, 1 yearearlier, 2 years earlier, 5 years earlier, or 10 years earlier, or more.

In some embodiments, the subject methods further include diagnosing anindividual as having a cognitive impairment, e.g., using the methodsdescribed herein or known in the art for measuring cognition andsynaptic plasticity, prior to administering the subjectplasma-comprising blood product. In some instances, the diagnosing willcomprise measuring cognition and/or synaptic plasticity and comparingthe results of the cognition or synaptic plasticity test to one or morereferences, e.g., a positive control and/or a negative control. Forexample, the reference may be the result(s) of the test performed by oneor more age-matched individuals that experience aging-associatedcognitive impairments (i.e., positive controls) or that do notexperience aging-associated cognitive impairments (i.e., negativecontrols). As another example, the reference may be the result(s) of thetest performed by the same individual at an earlier time, e.g., 2 weeksearlier, 1 month earlier, 2 months earlier, 3 months earlier, 6 monthsearlier, 1 year earlier, 2 years earlier, 5 years earlier, or 10 yearsearlier, or more.

In some embodiments, the subject methods further include diagnosing anindividual as having an aging-associated disorder, e.g., Alzheimer'sdisease, Parkinson's disease, frontotemporal dementia, progressivesupranuclear palsy, Huntington's disease, amyotrophic lateral sclerosis,spinal muscular atrophy, multiple sclerosis, multi-system atrophy,glaucoma, ataxias, muscular dystrophy, dementia, and the like. Methodsfor diagnosing such aging-associated disorders are well-known in theart, any of which may be used by the ordinarily skilled artisan indiagnosing the individual. In some embodiments, the subject methodsfurther comprise both diagnosing an individual as having anaging-associated disorder and as having a cognitive impairment.

Utility

The subject methods find use in treating, including preventing,aging-associated impairments and conditions associated therewith, suchas impairments in the cognitive ability of individuals. Individualssuffering from or at risk of developing an aging-associated cognitiveimpairments include individuals that are about 50 years old or older,e.g., 60 years old or older, 70 years old or older, 80 years old orolder, 90 years old or older, and usually no older than 100 years old,i.e., between the ages of about 50 and 100, e.g., 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or about 100 years old, and are suffering fromcognitive impairment associated with natural aging process, e.g., mildcognitive impairment (M.C.I.); and individuals that are about 50 yearsold or older, e.g., 60 years old or older, 70 years old or older, 80years old or older, 90 years old or older, and usually no older than 100years old, i.e., between the ages of about 50 and 90, e.g., 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or about 100 years old, that have not yetbegun to show symptoms of cognitive impairment. Examples of cognitiveimpairments that are due to natural aging include the following:

Mild cognitive impairment (M.C.I.) is a modest disruption of cognitionthat manifests as problems with memory or other mental functions such asplanning, following instructions, or making decisions that have worsenedover time while overall mental function and daily activities are notimpaired. Thus, although significant neuronal death does not typicallyoccur, neurons in the aging brain are vulnerable to sub-lethalage-related alterations in structure, synaptic integrity, and molecularprocessing at the synapse, all of which impair cognitive function.

Individuals suffering from or at risk of developing an aging-associatedcognitive impairment that will benefit from treatment with the subjectplasma-comprising blood product, e.g., by the methods disclosed herein,also include individuals of any age that are suffering from a cognitiveimpairment due to an aging-associated disorder; and individuals of anyage that have been diagnosed with an aging-associated disorder that istypically accompanied by cognitive impairment, where the individual hasnot yet begun to present with symptoms of cognitive impairment. Examplesof such aging-associated disorders include the following:

Alzheimer's Disease (AD).

Alzheimer's disease is a progressive, inexorable loss of cognitivefunction associated with an excessive number of senile plaques in thecerebral cortex and subcortical gray matter, which also containsβ-amyloid and neurofibrillary tangles consisting of tau protein. Thecommon form affects persons >60 yr old, and its incidence increases asage advances. It accounts for more than 65% of the dementias in theelderly.

The cause of Alzheimer's disease is not known. The disease runs infamilies in about 15 to 20% of cases. The remaining, so-called sporadiccases have some genetic determinants. The disease has an autosomaldominant genetic pattern in most early-onset and some late-onset casesbut a variable late-life penetrance. Environmental factors are the focusof active investigation.

In the course of the disease, synapses, and ultimately neurons are lostwithin the cerebral cortex, hippocampus, and subcortical structures(including selective cell loss in the nucleus basalis of Meynert), locuscaeruleus, and nucleus raphae dorsalis. Cerebral glucose use andperfusion is reduced in some areas of the brain (parietal lobe andtemporal cortices in early-stage disease, prefrontal cortex inlate-stage disease). Neuritic or senile plaques (composed of neurites,astrocytes, and glial cells around an amyloid core) and neurofibrillarytangles (composed of paired helical filaments) play a role in thepathogenesis of Alzheimer's disease. Senile plaques and neurofibrillarytangles occur with normal aging, but they are much more prevalent inpersons with Alzheimer's disease.

Parkinson's Disease.

Parkinson's Disease (PD) is an idiopathic, slowly progressive,degenerative CNS disorder characterized by slow and decreased movement,muscular rigidity, resting tremor, and postural instability. Originallyconsidered primarily a motor disorder, PD is now recognized to alsoaffect cognition, behavior, sleep, autonomic function, and sensoryfunction. The most common cognitive impairments include an impairment inattention and concentration, working memory, executive function,producing language, and visuospatial function.

In primary Parkinson's disease, the pigmented neurons of the substantianigra, locus caeruleus, and other brain stem dopaminergic cell groupsare lost. The cause is not known. The loss of substantia nigra neurons,which project to the caudate nucleus and putamen, results in depletionof the neurotransmitter dopamine in these areas. Onset is generallyafter age 40, with increasing incidence in older age groups.

Secondary parkinsonism results from loss of or interference with theaction of dopamine in the basal ganglia due to other idiopathicdegenerative diseases, drugs, or exogenous toxins. The most common causeof secondary parkinsonism is ingestion of antipsychotic drugs orreserpine, which produce parkinsonism by blocking dopamine receptors.Less common causes include carbon monoxide or manganese poisoning,hydrocephalus, structural lesions (tumors, infarcts affecting themidbrain or basal ganglia), subdural hematoma, and degenerativedisorders, including striatonigral degeneration.

Frontotemporal Dementia.

Frontotemporal dementia (FTD) is a condition resulting from theprogressive deterioration of the frontal lobe of the brain. Over time,the degeneration may advance to the temporal lobe. Second only toAlzheimer's disease (AD) in prevalence, FTD accounts for 20% ofpre-senile dementia cases. Symptoms are classified into three groupsbased on the functions of the frontal and temporal lobes affected:Behavioural variant FTD (bvFTD), with symptoms include lethargy andaspontaneity on the one hand, and disinhibition on the other;progressive nonfluent aphasia (PNFA), in which a breakdown in speechfluency due to articulation difficulty, phonological and/or syntacticerrors is observed but word comprehension is preserved; and semanticdementia (SD), in which patients remain fluent with normal phonology andsyntax but have increasing difficulty with naming and wordcomprehension. Other cognitive symptoms common to all FTD patientsinclude an impairment in executive function and ability to focus. Othercognitive abilities, including perception, spatial skills, memory andpraxis typically remain intact. FTD can be diagnosed by observation ofreveal frontal lobe and/or anterior temporal lobe atrophy in structuralMRI scans.

A number of forms of FTD exist, any of which may be treated or preventedusing the subject methods and compositions. For example, one form offrontotemporal dementia is Semantic Dementia (SD). SD is characterizedby a loss of semantic memory in both the verbal and non-verbal domains.SD patients often present with the complaint of word-findingdifficulties. Clinical signs include fluent aphasia, anomia, impairedcomprehension of word meaning, and associative visual agnosia (theinability to match semantically related pictures or objects). As thedisease progresses, behavioral and personality changes are often seensimilar to those seen in frontotemporal dementia although cases havebeen described of ‘pure’ semantic dementia with few late behavioralsymptoms. Structural MRI imaging shows a characteristic pattern ofatrophy in the temporal lobes (predominantly on the left), with inferiorgreater than superior involvement and anterior temporal lobe atrophygreater than posterior.

As another example, another form of frontotemporal dementia is Pick'sdisease (PiD, also PcD). A defining characteristic of the disease isbuild-up of tau proteins in neurons, accumulating into silver-staining,spherical aggregations known as “Pick bodies”. Symptoms include loss ofspeech (aphasia) and dementia. Patients with orbitofrontal dysfunctioncan become aggressive and socially inappropriate. They may steal ordemonstrate obsessive or repetitive stereotyped behaviors. Patients withdorsomedial or dorsolateral frontal dysfunction may demonstrate a lackof concern, apathy, or decreased spontaneity. Patients can demonstratean absence of self-monitoring, abnormal self-awareness, and an inabilityto appreciate meaning. Patients with gray matter loss in the bilateralposterolateral orbitofrontal cortex and right anterior insula maydemonstrate changes in eating behaviors, such as a pathologic sweettooth. Patients with more focal gray matter loss in the anterolateralorbitofrontal cortex may develop hyperphagia. While some of the symptomscan initially be alleviated, the disease progresses and patients oftendie within two to ten years.

Huntington's Disease.

Huntington's disease (HD) is a hereditary progressive neurodegenerativedisorder characterized by the development of emotional, behavioral, andpsychiatric abnormalities; loss of intellectual or cognitivefunctioning; and movement abnormalities (motor disturbances). Theclassic signs of HD include the development of chorea—involuntary,rapid, irregular, jerky movements that may affect the face, arms, legs,or trunk—as well as cognitive decline including the gradual loss ofthought processing and acquired intellectual abilities. There may beimpairment of memory, abstract thinking, and judgment; improperperceptions of time, place, or identity (disorientation); increasedagitation; and personality changes (personality disintegration).Although symptoms typically become evident during the fourth or fifthdecades of life, the age at onset is variable and ranges from earlychildhood to late adulthood (e.g., 70 s or 80 s).

HD is transmitted within families as an autosomal dominant trait. Thedisorder occurs as the result of abnormally long sequences or “repeats”of coded instructions within a gene on chromosome 4 (4p16.3). Theprogressive loss of nervous system function associated with HD resultsfrom loss of neurons in certain areas of the brain, including the basalganglia and cerebral cortex.

Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, invariablyfatal neurological disease that attacks motor neurons. Muscular weaknessand atrophy and signs of anterior horn cell dysfunction are initiallynoted most often in the hands and less often in the feet. The site ofonset is random, and progression is asymmetric. Cramps are common andmay precede weakness. Rarely, a patient survives 30 years; 50% diewithin 3 years of onset, 20% live 5 years, and 10% live 10 years.Diagnostic features include onset during middle or late adult life andprogressive, generalized motor involvement without sensoryabnormalities. Nerve conduction velocities are normal until late in thedisease. Recent studies have documented the presentation of cognitiveimpairments as well, particularly a reduction in immediate verbalmemory, visual memory, language, and executive function.

A decrease in cell body area, number of synapses and total synapticlength has been reported in even normal-appearing neurons of the ALSpatients. It has been suggested that when the plasticity of the activezone reaches its limit, a continuing loss of synapses can lead tofunctional impairment. Promoting the formation or new synapses orpreventing synapse loss may maintain neuron function in these patients.

Multiple Sclerosis.

Multiple Sclerosis (MS) is characterized by various symptoms and signsof CNS dysfunction, with remissions and recurring exacerbations. Themost common presenting symptoms are paresthesias in one or moreextremities, in the trunk, or on one side of the face; weakness orclumsiness of a leg or hand; or visual disturbances, e.g., partialblindness and pain in one eye (retrobulbar optic neuritis), dimness ofvision, or scotomas. Common cognitive impairments include impairments inmemory (acquiring, retaining, and retrieving new information), attentionand concentration (particularly divided attention), informationprocessing, executive functions, visuospatial functions, and verbalfluency. Common early symptoms are ocular palsy resulting in doublevision (diplopia), transient weakness of one or more extremities, slightstiffness or unusual fatigability of a limb, minor gait disturbances,difficulty with bladder control, vertigo, and mild emotionaldisturbances; all indicate scattered CNS involvement and often occurmonths or years before the disease is recognized. Excess heat mayaccentuate symptoms and signs.

The course is highly varied, unpredictable, and, in most patients,remittent. At first, months or years of remission may separate episodes,especially when the disease begins with retrobulbar optic neuritis.However, some patients have frequent attacks and are rapidlyincapacitated; for a few the course can be rapidly progressive.

Glaucoma.

Glaucoma is a common neurodegenerative disease that affects retinalganglion cells (RGCs). Evidence supports the existence ofcompartmentalized degeneration programs in synapses and dendrites,including in RGCs. Recent evidence also indicates a correlation betweencognitive impairment in older adults and glaucoma (Yochim B P, et al.Prevalence of cognitive impairment, depression, and anxiety symptomsamong older adults with glaucoma. J Glaucoma. 2012; 21(4):250-254).

Myotonic Dystrophy.

Myotonic dystrophy (DM) is an autosomal dominant multisystem disordercharacterized by dystrophic muscle weakness and myotonia. The moleculardefect is an expanded trinucleotide (CTG) repeat in the 3′ untranslatedregion of the myotonin-protein kinase gene on chromosome 19q. Symptomscan occur at any age, and the range of clinical severity is broad.Myotonia is prominent in the hand muscles, and ptosis is common even inmild cases. In severe cases, marked peripheral muscular weakness occurs,often with cataracts, premature balding, hatchet facies, cardiacarrhythmias, testicular atrophy, and endocrine abnormalities (e.g.,diabetes mellitus). Mental retardation is common in severe congenitalforms, while an aging-related decline of frontal and temporal cognitivefunctions, particularly language and executive functions, is observed inmilder adult forms of the disorder. Severely affected persons die bytheir early 50s.

Dementia.

Dementia describes class of disorders having symptoms affecting thinkingand social abilities severely enough to interfere with dailyfunctioning. Other instances of dementia in addition to the dementiaobserved in later stages of the aging-associated disorders discussedabove include vascular dementia, and dementia with Lewy bodies,described below.

In vascular dementia, or “multi-infarct dementia”, cognitive impairmentis caused by problems in supply of blood to the brain, typically by aseries of minor strokes, or sometimes, one large stroke preceded orfollowed by other smaller strokes. Vascular lesions can be the result ofdiffuse cerebrovascular disease, such as small vessel disease, or focallesions, or both. Patients suffering from vascular dementia present withcognitive impairment, acutely or subacutely, after an acutecerebrovascular event, after which progressive cognitive decline isobserved. Cognitive impairments are similar to those observed inAlzheimer's disease, including impairments in language, memory, complexvisual processing, or executive function, although the related changesin the brain are not due to AD pathology but to chronic reduced bloodflow in the brain, eventually resulting in dementia. Single photonemission computed tomography (SPECT) and positron emission tomography(PET) neuroimaging may be used to confirm a diagnosis of multi-infarctdementia in conjunction with evaluations involving mental statusexamination.

Dementia with Lewy bodies (DLB, also known under a variety of othernames including Lewy body dementia, diffuse Lewy body disease, corticalLewy body disease, and senile dementia of Lewy type) is a type ofdementia characterized anatomically by the presence of Lewy bodies(clumps of alpha-synuclein and ubiquitin protein) in neurons, detectablein post mortem brain histology. Its primary feature is cognitivedecline, particularly of executive functioning. Alertness and short termmemory will rise and fall. Persistent or recurring visual hallucinationswith vivid and detailed pictures are often an early diagnostic symptom.DLB it is often confused in its early stages with Alzheimer's diseaseand/or vascular dementia, although, where Alzheimer's disease usuallybegins quite gradually, DLB often has a rapid or acute onset. DLBsymptoms also include motor symptoms similar to those of Parkinson's.DLB is distinguished from the dementia that sometimes occurs inParkinson's disease by the time frame in which dementia symptoms appearrelative to Parkinson symptoms. Parkinson's disease with dementia (PDD)would be the diagnosis when dementia onset is more than a year after theonset of Parkinson's. DLB is diagnosed when cognitive symptoms begin atthe same time or within a year of Parkinson symptoms.

Progressive Supranuclear Palsy.

Progressive supranuclear palsy (PSP) is a brain disorder that causesserious and progressive problems with control of gait and balance, alongwith complex eye movement and thinking problems. One of the classicsigns of the disease is an inability to aim the eyes properly, whichoccurs because of lesions in the area of the brain that coordinates eyemovements. Some individuals describe this effect as a blurring. Affectedindividuals often show alterations of mood and behavior, includingdepression and apathy as well as progressive mild dementia. Thedisorders long name indicates that the disease begins slowly andcontinues to get worse (progressive), and causes weakness (palsy) bydamaging certain parts of the brain above pea-sized structures callednuclei that control eye movements (supranuclear). PSP was firstdescribed as a distinct disorder in 1964, when three scientistspublished a paper that distinguished the condition from Parkinson'sdisease. It is sometimes referred to as Steele-Richardson-Olszewskisyndrome, reflecting the combined names of the scientists who definedthe disorder. Although PSP gets progressively worse, no one dies fromPSP itself.

Ataxia.

People with ataxia have problems with coordination because parts of thenervous system that control movement and balance are affected. Ataxiamay affect the fingers, hands, arms, legs, body, speech, and eyemovements. The word ataxia is often used to describe a symptom ofincoordination which can be associated with infections, injuries, otherdiseases, or degenerative changes in the central nervous system. Ataxiais also used to denote a group of specific degenerative diseases of thenervous system called the hereditary and sporadic ataxias which are theNational Ataxia Foundation's primary emphases.

Multiple-System Atrophy.

Multiple-system atrophy (MSA) is a degenerative neurological disorder.MSA is associated with the degeneration of nerve cells in specific areasof the brain. This cell degeneration causes problems with movement,balance, and other autonomic functions of the body such as bladdercontrol or blood-pressure regulation. The cause of MSA is unknown and nospecific risk factors have been identified. Around 55% of cases occur inmen, with typical age of onset in the late 50s to early 60s. MSA oftenpresents with some of the same symptoms as Parkinson's disease. However,MSA patients generally show minimal if any response to the dopaminemedications used for Parkinson's.

In some embodiments, the subject methods and compositions find use inslowing the progression of aging-associated cognitive impairment. Inother words, cognitive abilities in the individual will decline moreslowly following treatment by the disclosed methods than prior to or inthe absence of treatment by the disclosed methods. In some suchinstances, the subject methods of treatment include measuring theprogression of cognitive decline after treatment, and determining thatthe progression of cognitive decline is reduced. In some such instances,the determination is made by comparing to a reference, e.g., the rate ofcognitive decline in the individual prior to treatment, e.g., asdetermined by measuring cognition prior at two or more time points priorto administration of the subject blood product.

The subject methods and compositions also find use in stabilizing thecognitive abilities of an individual, e.g., an individual suffering fromaging-associated cognitive decline or an individual at risk of sufferingfrom aging-associated cognitive decline. For example, the individual maydemonstrate some aging-associated cognitive impairment, and progressionof cognitive impairment observed prior to treatment with the disclosedmethods will be halted following treatment by the disclosed methods. Asanother example, the individual may be at risk for developing anaging-associated cognitive decline (e.g., the individual may be aged 50years old or older, or may have been diagnosed with an aging-associateddisorder), and the cognitive abilities of the individual aresubstantially unchanged, i.e., no cognitive decline can be detected,following treatment by the disclosed methods as compared to prior totreatment with the disclosed methods.

The subject methods and compositions also find use in reducing cognitiveimpairment in an individual suffering from an aging-associated cognitiveimpairment. In other words, cognitive ability is improved in theindividual following treatment by the subject methods. For example, thecognitive ability in the individual is increased, e.g., by 2-fold ormore, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more,30-fold or more, or 40-fold or more, including 50-fold or more, 60-foldor more, 70-fold or more, 80-fold or more, 90-fold or more, or 100-foldor more, following treatment by the subject methods relative to thecognitive ability that is observed in the individual prior to treatmentby the subject methods. In some instances, treatment by the subjectmethods and compositions restores the cognitive ability in theindividual suffering from aging-associated cognitive decline, e.g., totheir level when the individual was about 40 years old or less. In otherwords, cognitive impairment is abrogated.

Combination Therapies

Active agents of the invention can be administered to a subject alone orin combination with an additional, i.e., second, active agent. As such,in some cases, the subject method further comprises administering to thesubject at least one additional compound. Any convenient agents may beutilized. For example, TIMP active agents can be supplied alone or inconjunction with one or more other drugs, such as drugs employed in thetreatment of aging associated conditions, e.g., cholinesteraseinhibitors (e.g., Donepezil, Rivastigmine, Galantamine, Tacrine),Memantine, Vitamin E, citalopram (Celexa), fluoxetine (Prozac),paroxeine (Paxil), sertraline (Zoloft), trazodone (Desyrel), lorazepam(Ativan), oxazepam (Serax), aripiprazole (Abilify), clozapine(Clozaril), haloperidol (Haldol), olanzapine (Zyprexa), quetiapine(Seroquel), risperidone (Risperdal), and ziprasidone (Geodon); non-TIMPpolypeptide active agents; e.g., chemokine (C—C motif) ligand 2 (CCL2)(i.e., MCP1); C—C motif chemokine 11 (i.e., chemotactic protein oreotaxin-1); Granulocyte-macrophage colony-stimulating factor(GM-CSF)(i.e., colony stimulating factor 2 or CSF2); etc.

The terms “co-administration” and “in combination with” include theadministration of two or more therapeutic agents either simultaneously,concurrently or sequentially within no specific time limits. In oneembodiment, the agents are present in the cell or in the subject's bodyat the same time or exert their biological or therapeutic effect at thesame time. In one embodiment, the therapeutic agents are in the samecomposition or unit dosage form. In other embodiments, the therapeuticagents are in separate compositions or unit dosage forms. In certainembodiments, a first agent can be administered prior to (e.g., minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapeutic agent.

“Concomitant administration” of a known therapeutic drug with apharmaceutical composition of the present invention means administrationof the drug and nucleoside agent at such time that both the known drugand the composition of the present invention will have a therapeuticeffect. Such concomitant administration may involve concurrent (i.e. atthe same time), prior, or subsequent administration of the drug withrespect to the administration of a subject nucleoside agent. Routes ofadministration of the two agents may vary, where representative routesof administration are described in greater detail below. A person ofordinary skill in the art would have no difficulty determining theappropriate timing, sequence and dosages of administration forparticular drugs and nucleoside agents of the present invention.

In some embodiments, the compounds are administered to the subjectwithin twenty-four hours of each other, such as within 12 hours of eachother, within 6 hours of each other, within 3 hours of each other, orwithin 1 hour of each other. In certain embodiments, the compounds areadministered within 1 hour of each other. In certain embodiments, thecompounds are administered substantially simultaneously. By administeredsubstantially simultaneously is meant that the compounds areadministered to the subject within about 10 minutes or less of eachother, such as 5 minutes or less, or 1 minute or less of each other.

Pharmaceutical Preparations

Also provided are pharmaceutical preparations of the subject compounds.The subject compounds can be incorporated into a variety of formulationsfor administration to a subject. More particularly, the compounds of thepresent invention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.The formulations may be designed for administration via a number ofdifferent routes, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginic acid;binding agents, for example starch, gelatin or acacia, and lubricatingagents, for example, magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed. They may also be coated by the technique described inthe U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredients is mixed with water oran oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethyl-cellulose, methylcellulose, hydroxy-propylmethycellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The compounds can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

The compounds of this invention and their pharmaceutically acceptablesalts which are active on topical administration can be formulated astransdermal compositions or transdermal delivery devices (“patches”).Such compositions include, for example, a backing, active compoundreservoir, a control membrane, liner and contact adhesive. Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference in its entirety. Such patches may be constructed forcontinuous, pulsatile, or on demand delivery of pharmaceutical agents.

Optionally, the pharmaceutical composition may contain otherpharmaceutically acceptable components, such a buffers, surfactants,antioxidants, viscosity modifying agents, preservatives and the like.Each of these components is well-known in the art. See, for example,U.S. Pat. No. 5,985,310, the disclosure of which is herein incorporatedby reference.

Other components suitable for use in the formulations of the presentinvention can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). In anembodiment, the aqueous cyclodextrin solution further comprise dextrose,e.g., about 5% dextrose.

Dosage levels of the order of from about 0.01 mg to about 140 mg/kg ofbody weight per day are useful in representative embodiments, oralternatively about 0.5 mg to about 7 g per patient per day. Forexample, inflammation may be effectively treated by the administrationof from about 0.01 to 50 mg of the compound per kilogram of body weightper day, or alternatively about 0.5 mg to about 3.5 g per patient perday. Those of skill will readily appreciate that dose levels can vary asa function of the specific compound, the severity of the symptoms andthe susceptibility of the subject to side effects. Dosages for a givencompound are readily determinable by those of skill in the art by avariety of means.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration of humans may containfrom 0.5 mg to 5 g of active agent compounded with an appropriate andconvenient amount of carrier material which may vary from about 5 toabout 95 percent of the total composition. Dosage unit forms willgenerally contain between from about 1 mg to about 500 mg of an activeingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500mg, 600 mg, 800 mg, or 1000 mg.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theage, body weight, general health, sex, diet, time of administration,route of administration, rate of excretion, drug combination and theseverity of the particular disease undergoing therapy.

As such, unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore inhibitors. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibitor(s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier. The term “unit dosage form,” asused herein, refers to physically discrete units suitable as unitarydosages for human and animal subjects, each unit containing apredetermined quantity of compounds of the present invention calculatedin an amount sufficient to produce the desired effect in associationwith a pharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the novel unit dosage forms of the present inventiondepend on the particular peptidomimetic compound employed and the effectto be achieved, and the pharmacodynamics associated with each compoundin the host.

Kits & Systems

Also provided are kits and systems that find use in practicingembodiments of the methods, such as those described as described above.The term “system” as employed herein refers to a collection of two ormore different active agents, present in a single composition or indisparate compositions, that are brought together for the purpose ofpracticing the subject methods. The term “kit” refers to a packagedactive agent or agents. For example, kits and systems for practicing thesubject methods may include one or more pharmaceutical formulations. Assuch, in certain embodiments the kits may include a singlepharmaceutical composition, present as one or more unit dosages, wherethe composition may include one or more expression/activity inhibitorcompounds. In yet other embodiments, the kits may include two or moreseparate pharmaceutical compositions, each containing a different activecompound.

Also of interest are kits and systems finding use in assays of theinvention, e.g., as described above. Such kits and systems may includeone or more components of the assays, e.g., vectors encoding fusionproteins, enzyme substrates, buffers, etc.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, portable flash drive, etc., on which the informationhas been recorded. Yet another means that may be present is a websiteaddress which may be used via the internet to access the information ata removed site. Any convenient means may be present in the kits.

The following examples are provided by way of illustration and not byway of limitation.

EXPERIMENTAL

Recombinant TIMP2 protein was delivered at a concentration of 50 μg/kgfour times over the course of a week by intraperitoneal injection inaged wildtype (C57Bl/6J) mice. Brains of treated mice revealed elevatedlevels of active neurons, namely those expressing the immediate earlygene c-Fos in the dentate gyrus subregion of the hippocampus. Eight(long-term) intraperitoneal injections (50 μg/kg) of TIMP2 were given toaged wildtype mice every other day prior to Barnes maze, nesting, andfear conditioning assessment. Behavioral testing revealed significantlyimproved performance in all three tasks in TIMP2-treated mice. Seven(long-term) intraperitoneal injections (50 μg/kg) of TIMP2 were given toaged wildtype mice every other day alone or in combination with anothercognition-promoting factor, CSF2. Brains of TIMP2 and TIMP2+CSF2 micerevealed significantly elevated levels of c-Fos+ (active) neurons in thedentate gyrus.

The results demonstrated that TIMP2 was sufficient to elevate the numberof c-Fos-expressing cells in the hippocampus of aged mice. Whenadministered systemically over a longer timecourse, TIMP2 is sufficientto increase the number of c-Fos-expressing cells, reverse learning andmemory deficits in several behavioral paradigms, and restore impairmentsin nesting ability. The above results demonstrate that TIMP2 is suitableto provide cognitive (learning and memory) benefits to patientssuffering from age-related cognitive impairments or neurodegenerativediseases that decrease synaptic function, e.g. Alzheimer's disease. Ourdiscovery represents the first description of a protein (TIMP2) thatdeclines with age in blood that could be used to reverse impairments insynaptic plasticity and age-related learning and memory deficits. TIMP2,when administered systemically for a period of only ˜2 weeks, is able toconfer enhanced plasticity and memory and learning function without theneed for direct to delivery to the brain.

Systemic TIMP2 treatment in aged mice robustly improves long-termpotentiation, a cellular correlate of learning and memory. Brain slicesisolated from aged wildtype mice that were treated with recombinantTIMP2 (i.p., 50 μg/kg) display enhanced long-term potentiation (LTP) ascompared to a control (vehicle). Shown in FIG. 1A are population spikeamplitudes (PSA) within dentate gyrus following stimulation in theperforant path of the hippocampus. Quantification of the maintenancephase of the PSA shown in FIG. 1A is provided in FIG. 1B. (Mean+/−SEM;Student's t test; *P<0.05.)

Systemic TIMP2 is necessary for hippocampal-dependent spatial memory, asassessed in a novel location recognition task. TIMP2 levels in youngwildtype mice were targeted for approximately one month using anantibody-mediated neutralization approach (60 μg/kg) prior to assessmentof object location displacement discrimination 24 hours after training.(Mean+/−SEM; 2-way ANOVA, followed by Tukey's post hoc test;****P<0.0001.) The results are shown in FIG. 2.

There are currently no effective treatments for the significant declinein synaptic plasticity, learning/memory, and other cognitive abilityassociated with normal brain aging or neurodegeneration. We haveidentified a youth-derived protein, TIMP2, with strong rejuvenatingactivity. TIMP2, when administered systemically for a relatively shortcourse of treatment, is able to confer enhanced plasticity and memoryand learning function without the need for direct delivery to the brain.TIMP2 is not a growth factor, nor is it a canonical immune signalingmolecule, which highlights an additional advantage, namely that TIMP2affords the ability to target brain aging processes without supplying apotentially tumorigenic or proinflammatory molecule that may harm otherorgan systems. Moreover, utilizing a protein naturally produced andfound in blood to limit cognitive dysfunction is unlikely to produceharmful side effects compared to conventional small molecule drugdesign.

No effective therapeutic agents exist to treat the age-related outcomesof CNS aging. Our approach allows for facile, systemic treatment ofelderly patients or those otherwise suffering from cognitive impairmentwith TIMP2, a protein normally produced by the body.

Notwithstanding the appended clauses, the disclosure is also defined bythe following clauses:

1. A method of treating an adult mammal for an aging-associatedcondition, the method comprising:

enhancing a TIMP activity in the mammal in a manner sufficient to treatthe adult mammal for the aging-associated condition.

2. The method according to Clause 1, wherein the TIMP activity is aTIMP1, TIMP2, TIMP3 or TIMP4 activity.

3. The method according to Clause 2, wherein the TIMP activity is aTIMP2 activity.

4. The method according to any of Clauses 1 to 3, where the methodcomprises enhancing a systemic TIMP activity.

5. The method according to any of Clauses 1 to 4, wherein the methodcomprises increasing a systemic level of a TIMP active agent in themammal.

6. The method according to Clause 5, wherein the systemic level of aTIMP active agent is increased by administering a TIMP active agent tothe mammal.

7. The method according to Clause 6, wherein the TIMP active agent is aTIMP polypeptide or mimetic thereof.

8. The method according to Clause 7, wherein the TIMP active agent is aTIMP polypeptide.

9. The method according to Clause 8, wherein the TIMP polypeptide has asequence that is at least 60% identical to any of SEQ ID NOS: 1 to 4.

10. The method according to any of clauses 1 to 5, wherein the methodcomprises enhancing expression of an endogenous TIMP coding sequence.

11. The method according to Clauses 1 to 4, wherein the method comprisespotentiating TIMP in the subject.

12. The method according to any of the preceding clauses, wherein themammal is a primate.

13. The method according to Clause 12, wherein the primate is a human.

14. The method according to any of the preceding clauses, wherein theadult mammal is an elderly mammal.

15. The method according to Clause 14, wherein the elderly mammal is ahuman that is 60 years or older.

16. The method according to any of the preceding clauses, wherein theaging-associated condition comprises a cognitive impairment.

17. The method according to any of the preceding clauses, wherein theadult mammal suffers from an aging associated disease condition.

18. The method according to any of the preceding clauses, wherein theaging associated disease condition is a cognitive decline diseasecondition.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

That which is claimed is:
 1. A method of ameliorating a symptom ofaging-associated cognitive impairment or decline in an adult mammal, themethod comprising: administering a Tissue inhibitor of metalloproteinase2 (TIMP2) polypeptide or a nucleic acid sequence encoding a TIMP2polypeptide to the mammal, thereby enhancing a TIMP2 activity in themammal in a manner sufficient to ameliorate a symptom ofaging-associated cognitive impairment or decline.
 2. The methodaccording to claim 1, where the method comprises increasing a systemiclevel of a TIMP2 polypeptide in the adult mammal.
 3. The methodaccording to claim 1, wherein the TIMP2 polypeptide has a sequenceidentical to SEQ ID NO:01.
 4. The method according to claim 1, whereinthe mammal is a primate.
 5. The method according to claim 4, wherein theprimate is a human.
 6. The method according to claim 5, wherein theprimate is greater than 50 years old.
 7. The method according to claim1, wherein the aging-associated cognitive impairment or decline iscognitive impairment.
 8. The method according to claim 1, wherein theaging-associated cognitive impairment or decline is cognitive decline.